MYC regulates fatty acid metabolism through a multigenic program in claudin-low triple negative breast cancer.

BACKGROUND: Recent studies have suggested that fatty acid oxidation (FAO) is a key metabolic pathway for the growth of triple negative breast cancers (TNBCs), particularly those that have high expression of MYC. However, the underlying mechanism by which MYC promotes FAO remains poorly understood. METHODS: We used a combination of metabolomics, transcriptomics, bioinformatics, and microscopy to elucidate a potential mechanism by which MYC regulates FAO in TNBC. RESULTS: We propose that MYC induces a multigenic program that involves changes in intracellular calcium signalling and fatty acid metabolism. We determined key roles for fatty acid transporters (CD36), lipases (LPL), and kinases (PDGFRB, CAMKK2, and AMPK) that each contribute to promoting FAO in human mammary epithelial cells that express oncogenic levels of MYC. Bioinformatic analysis further showed that this multigenic program is highly expressed and predicts poor survival in the claudin-low molecular subtype of TNBC, but not other subtypes of TNBCs, suggesting that efforts to target FAO in the clinic may best serve claudin-low TNBC patients. CONCLUSION: We identified critical pieces of the FAO machinery that have the potential to be targeted for improved treatment of patients with TNBC, especially the claudin-low molecular subtype.

Age decreases mitochondrial motility and increases mitochondrial size in vascular smooth muscle.

KEY POINTS: Age is proposed to be associated with altered structure and function of mitochondria; however, in fully-differentiated cells, determining the structure of more than a few mitochondria at a time is challenging. In the present study, the structures of the entire mitochondrial complements of cells were resolved from a pixel-by-pixel covariance analysis of fluctuations in potentiometric fluorophore intensity during 'flickers' of mitochondrial membrane potential. Mitochondria are larger in vascular myocytes from aged rats compared to those in younger adult rats. A subpopulation of mitochondria in myocytes from aged, but not younger, animals is highly-elongated. Some mitochondria in myocytes from younger, but not aged, animals are highly-motile. Mitochondria that are motile are located more peripherally in the cell than non-motile mitochondria. ABSTRACT: Mitochondrial function, motility and architecture are each central to cell function. Age-associated mitochondrial dysfunction may contribute to vascular disease. However, mitochondrial changes in ageing remain ill-defined because of the challenges of imaging in native cells. We determined the structure of mitochondria in live native cells, demarcating boundaries of individual organelles by inducing stochastic 'flickers' of membrane potential, recorded as fluctuations in potentiometric fluorophore intensity (flicker-assisted localization microscopy; FaLM). In freshly-isolated myocytes from rat cerebral resistance arteries, FaLM showed a range of mitochondrial X-Y areas in both young adult (3 months; 0.05-6.58 µm(2) ) and aged rats (18 months; 0.05-13.4 µm(2) ). In cells from young animals, most mitochondria were small (mode area 0.051 µm(2) ) compared to aged animals (0.710 µm(2) ). Cells from older animals contained a subpopulation of highly-elongated mitochondria (5.3% were >2 µm long, 4.2% had a length:width ratio >3) that was rare in younger animals (0.15% of mitochondria >2 µm long, 0.4% had length:width ratio >3). The extent of mitochondrial motility also varied. 1/811 mitochondria observed moved slightly (∼0.5 µm) in myocytes from older animals, whereas, in the younger animals, directed and Brownian-like motility occurred regularly (215 of 1135 mitochondria moved within 10 min, up to distance of 12 µm). Mitochondria positioned closer to the cell periphery showed a greater tendency to move. In conclusion, cerebral vascular myocytes from young rats contained small, motile mitochondria. In aged rats, mitochondria were larger, immobile and could be highly-elongated. These age-associated alterations in mitochondrial behaviour may contribute to alterations in cell signalling, energy supply or the onset of proliferation.

Synthesis of an azido-tagged low affinity ratiometric calcium sensor.

Changes in high localised concentrations of Ca2+ ions are fundamental to cell signalling. The synthesis of a dual excitation, ratiometric calcium ion sensor with a Kd of 90 µM, is described. It is tagged with an azido group for bioconjugation, and absorbs in the blue/green and emits in the red region of the visible spectrum with a large Stokes shift. The binding modulating nitro group is introduced to the BAPTA core prior to construction of a benzofuran-2-yl carboxaldehyde by an allylation-oxidation-cyclisation sequence, which is followed by condensation with an azido-tagged thiohydantoin. The thiohydantoin unit has to be protected with an acetoxymethyl (AM) caging group to allow CuAAC click reaction and incorporation of the KDEL peptide endoplasmic reticulum (ER) retention sequence.

Microdomains of muscarinic acetylcholine and Ins(1,4,5)P3 receptors create 'Ins(1,4,5)P3 junctions' and sites of Ca²+ wave initiation in smooth muscle.

Increases in cytosolic Ca(2+) concentration ([Ca(2+)](c)) mediated by inositol (1,4,5)-trisphosphate [Ins(1,4,5)P(3), hereafter InsP(3)] regulate activities that include division, contraction and cell death. InsP(3)-evoked Ca(2+) release often begins at a single site, then regeneratively propagates through the cell as a Ca(2+) wave. The Ca(2+) wave consistently begins at the same site on successive activations. Here, we address the mechanisms that determine the Ca(2+) wave initiation site in intestinal smooth muscle cells. Neither an increased sensitivity of InsP(3) receptors (InsP(3)R) to InsP(3) nor regional clustering of muscarinic receptors (mAChR3) or InsP(3)R1 explained the selection of an initiation site. However, examination of the overlap of mAChR3 and InsP(3)R1 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 Ca(2+) wave initiation site. The initiation site might arise from a selective delivery of InsP(3) from mAChR3 activity to particular InsP(3)Rs to generate faster local [Ca(2+)](c) increases at sites of colocalisation. In support of this hypothesis, a localised subthreshold 'priming' InsP(3) concentration applied rapidly, but at regions distant from the initiation site, shifted the wave to the site of the priming. Conversely, when the Ca(2+) rise at the initiation site was rapidly and selectively attenuated, the Ca(2+) wave again shifted and initiated at a new site. These results indicate that Ca(2+) waves initiate where there is a structural and functional coupling of mAChR3 and InsP(3)R1, which generates junctions in which InsP(3) acts as a highly localised signal by being rapidly and selectively delivered to InsP(3)R1.

Examining the role of mitochondria in Ca²âº signaling in native vascular smooth muscle.

Mitochondrial Ca²âº uptake contributes important feedback controls to limit the time course of Ca²âº signals. Mitochondria regulate cytosolic [Ca²âº] over an exceptional breath of concentrations (~200 nM to >10 µM) to provide a wide dynamic range in the control of Ca²âº signals. Ca²âº 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 Ca²âº 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 Ca²âº 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.

Mitochondrial motility and vascular smooth muscle proliferation.

OBJECTIVE: Mitochondria are widely described as being highly dynamic and adaptable organelles, and their movement is thought to be vital for cell function. Yet, in various native cells, including those of heart and smooth muscle, mitochondria are stationary and rigidly structured. The significance of the differences in mitochondrial behavior to the physiological function of cells is unclear and was studied in single myocytes and intact resistance-sized cerebral arteries. We hypothesized that mitochondrial dynamics is controlled by the proliferative status of the cells. METHODS AND RESULTS: High-speed fluorescence imaging of mitochondria in live vascular smooth muscle cells shows that the organelle undergoes significant reorganization as cells become proliferative. In nonproliferative cells, mitochondria are individual (≈ 2 µm by 0.5 µm), stationary, randomly dispersed, fixed structures. However, on entering the proliferative state, mitochondria take on a more diverse architecture and become small spheres, short rod-shaped structures, long filamentous entities, and networks. When cells proliferate, mitochondria also continuously move and change shape. In the intact pressurized resistance artery, mitochondria are largely immobile structures, except in a small number of cells in which motility occurred. When proliferation of smooth muscle was encouraged in the intact resistance artery, in organ culture, the majority of mitochondria became motile and the majority of smooth muscle cells contained moving mitochondria. Significantly, restriction of mitochondrial motility using the fission blocker mitochondrial division inhibitor prevented vascular smooth muscle proliferation in both single cells and the intact resistance artery. CONCLUSIONS: These results show that mitochondria are adaptable and exist in intact tissue as both stationary and highly dynamic entities. This mitochondrial plasticity is an essential mechanism for the development of smooth muscle proliferation and therefore presents a novel therapeutic target against vascular disease.

Mitochondrial regulation of cytosolic Ca²âº signals in smooth muscle.

The cytosolic Ca²âº concentration ([Ca²âº]c) controls virtually every activity of smooth muscle, including contraction, migration, transcription, division and apoptosis. These processes may be activated by large (>10 µM) amplitude [Ca²âº]c increases, which occur in small restricted regions of the cell or by smaller (<1 µM) amplitude changes throughout the bulk cytoplasm. Mitochondria contribute to the regulation of these signals by taking up Ca²âº. However, mitochondria's reported low affinity for Ca²âº is thought to require the organelle to be positioned close to ion channels and within a microdomain of high [Ca²âº]. In cultured smooth muscle, mitochondria are highly dynamic structures but in native smooth muscle mitochondria are immobile, apparently strategically positioned organelles that regulate the upstroke and amplitude of IP3-evoked Ca²âº signals and IP3 receptor (IP3R) cluster activity. These observations suggest mitochondria are positioned within the high [Ca²âº] microdomain arising from an IP3R cluster to exert significant local control of channel activity. On the other hand, neither the upstroke nor amplitude of voltage-dependent Ca²âº entry is modulated by mitochondria; rather, it is the declining phase of the transient that is regulated by the organelle. Control of the declining phase of the transient requires a high mitochondrial affinity for Ca²âº to enable uptake to occur over the normal physiological Ca²âº range (<1 µM). Thus, in smooth muscle, mitochondria regulate Ca²âº signals exerting effects over a large range of [Ca²âº] (∼200 nM to at least tens of micromolar) to provide a wide dynamic range in the control of Ca²âº signals.

Selective uncoupling of individual mitochondria within a cell using a mitochondria-targeted photoactivated protonophore.

Depolarization of an individual mitochondrion or small clusters of mitochondria within cells has been achieved using a photoactivatable probe. The probe is targeted to the matrix of the mitochondrion by an alkyltriphenylphosphonium lipophilic cation and releases the protonophore 2,4-dinitrophenol locally in predetermined regions in response to directed irradiation with UV light via a local photolysis system. This also provides a proof of principle for the general temporally and spatially controlled release of bioactive molecules, pharmacophores, or toxins to mitochondria with tissue, cell, or mitochondrion specificity.

Subplasma membrane Ca2+ signals.

Ca(2+) 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 Ca(2+) 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 Ca(2+) signals near the plasma membrane, but probably the most prevalent involve the use of fluorescent Ca(2+) indicators and fall into two general approaches. In the first, the Ca(2+) indicators themselves are specifically targeted to the subplasma membrane space to measure Ca(2+) only there. Alternatively, the indicators are allowed to be dispersed throughout the cytoplasm, but the fluorescence emanating from the Ca(2+) 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 Ca(2+) are a shortcoming. Use of targeted indicators is also largely restricted to cultured cells. High resolution imaging applied with rapidly responding small molecule Ca(2+) 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 Ca(2+) signals and methods for their measurement is provided.

Mitochondrial organization and Ca2+ uptake.

Mitochondria may function as multiple separate organelles or as a single electrically coupled continuum to modulate changes in [Ca2+]c (cytoplasmic Ca2+ concentration) in various cell types. Mitochondria may also be tethered to the internal Ca2+ store or plasma membrane in particular parts of cells to facilitate the organelles modulation of local and global [Ca2+]c increases. Differences in the organization and positioning contributes significantly to the at times apparently contradictory reports on the way mitochondria modulate [Ca2+]c signals. In the present paper, we review the organization of mitochondria and the organelles role in Ca2+ signalling.

Mitochondrial Ca2+ uptake increases Ca2+ release from inositol 1,4,5-trisphosphate receptor clusters in smooth muscle cells.

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.

Agonist-evoked Ca(2+) wave progression requires Ca(2+) and IP(3).

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

Sunitinib and Imatinib Display Differential Cardiotoxicity in Adult Rat Cardiac Fibroblasts That Involves a Role for Calcium/Calmodulin Dependent Protein Kinase II.

Background: Tyrosine kinase inhibitors (TKIs) have dramatically improved cancer treatment but are known to cause cardiotoxicity. The pathophysiological consequences of TKI therapy are likely to manifest across different cell types of the heart, yet there is little understanding of the differential adverse cellular effects. Cardiac fibroblasts (CFs) play a pivotal role in the repair and remodeling of the heart following insult or injury, yet their involvement in anti-cancer drug induced cardiotoxicity has been largely overlooked. Here, we examine the direct effects of sunitinib malate and imatinib mesylate on adult rat CF viability, Ca2+ handling and mitochondrial function that may contribute to TKI-induced cardiotoxicity. In particular, we investigate whether Ca2+/calmodulin dependent protein kinase II (CaMKII), may be a mediator of TKI-induced effects. Methods: CF viability in response to chronic treatment with both drugs was assessed using MTT assays and flow cytometry analysis. Calcium mobilization was assessed in CFs loaded with Fluo4-AM and CaMKII activation via oxidation was measured via quantitative immunoblotting. Effects of both drugs on mitochondrial function was determined by live mitochondrial imaging using MitoSOX red. Results: Treatment of CFs with sunitinib (0.1-10 µM) resulted in concentration-dependent alterations in CF phenotype, with progressively significant cell loss at higher concentrations. Flow cytometry analysis and MTT assays revealed increased cell apoptosis and necrosis with increasing concentrations of sunitinib. In contrast, equivalent concentrations of imatinib resulted in no significant change in cell viability. Both sunitinib and imatinib pre-treatment increased Angiotensin II-induced intracellular Ca2+ mobilization, with only sunitinib resulting in a significant effect and also causing increased CaMKII activation via oxidation. Live cell mitochondrial imaging using MitoSOX red revealed that both sunitinib and imatinib increased mitochondrial superoxide production in a concentration-dependent manner. This effect in response to both drugs was suppressed in the presence of the CaMKII inhibitor KN-93. Conclusions: Sunitinib and imatinib showed differential effects on CFs, with sunitinib causing marked changes in cell viability at concentrations where imatinib had no effect. Sunitinib caused a significant increase in Angiotensin II-induced intracellular Ca2+ mobilization and both TKIs caused increased mitochondrial superoxide production. Targeted CaMKII inhibition reversed the TKI-induced mitochondrial damage. These findings highlight a new role for CaMKII in TKI-induced cardiotoxicity, particularly at the level of the mitochondria, and confirm differential off-target toxicity in CFs, consistent with the differential selectivity of sunitinib and imatinib.

Multi-Omics Studies Demonstrate Toxoplasma gondii-Induced Metabolic Reprogramming of Murine Dendritic Cells.

Toxoplasma gondii is capable of actively invading almost any mammalian cell type including phagocytes. Early events in phagocytic cells such as dendritic cells are not only key to establishing parasite infection, but conversely play a pivotal role in initiating host immunity. It is now recognized that in addition to changes in canonical immune markers and mediators, alteration in metabolism occurs upon activation of phagocytic cells. These metabolic changes are important for supporting the developing immune response, but can affect the availability of nutrients for intracellular pathogens including T. gondii. However, the interaction of T. gondii with these cells and particularly how infection changes their metabolism has not been extensively investigated. Herein, we use a multi-omics approach comprising transcriptomics and metabolomics validated with functional assays to better understand early events in these cells following infection. Analysis of the transcriptome of T. gondii infected bone marrow derived dendritic cells (BMDCs) revealed significant alterations in transcripts associated with cellular metabolism, activation of T cells, inflammation mediated chemokine and cytokine signaling pathways. Multivariant analysis of metabolomic data sets acquired through non-targeted liquid chromatography mass spectroscopy (LCMS) identified metabolites associated with glycolysis, the TCA cycle, oxidative phosphorylation and arginine metabolism as major discriminants between control uninfected and T. gondii infected cells. Consistent with these observations, glucose uptake and lactate dehydrogenase activity were upregulated in T. gondii infected BMDC cultures compared with control BMDCs. Conversely, BMDC mitochondrial membrane potential was reduced in T. gondii-infected cells relative to mitochondria of control BMDCs. These changes to energy metabolism, similar to what has been described following LPS stimulation of BMDCs and macrophages are often termed the Warburg effect. This metabolic reprogramming of cells has been suggested to be an important adaption that provides energy and precursors to facilitate phagocytosis, antigen processing and cytokine production. Other changes to BMDC metabolism are evident following T. gondii infection and include upregulation of arginine degradation concomitant with increased arginase-1 activity and ornithine and proline production. As T. gondii is an arginine auxotroph the resultant reduced cellular arginine levels are likely to curtail parasite multiplication. These results highlight the complex interplay of BMDCs and parasite metabolism within the developing immune response and the consequences for adaptive immunity and pathogen clearance.

RPGR protein complex regulates proteasome activity and mediates store-operated calcium entry.

Ciliopathies are a group of genetically heterogeneous disorders, characterized by defects in cilia genesis or maintenance. Mutations in the RPGR gene and its interacting partners, RPGRIP1 and RPGRIP1L, cause ciliopathies, but the function of their proteins remains unclear. Here we show that knockdown (KD) of RPGR, RPGRIP1 or RPGRIP1L in hTERT-RPE1 cells results in abnormal actin cytoskeleton organization. The actin cytoskeleton rearrangement is regulated by the small GTPase RhoA via the planar cell polarity (PCP) pathway. RhoA activity was upregulated in the absence of RPGR, RPGRIP1 or RPGRIP1L proteins. In RPGR, RPGRIP1 or RPGRIP1L KD cells, we observed increased levels of DVl2 and DVl3 proteins, the core components of the PCP pathway, due to impaired proteasomal activity. RPGR, RPGRIP1 or RPGRIP1L KD cells treated with thapsigargin (TG), an inhibitor of sarcoendoplasmic reticulum Ca2+- ATPases, showed impaired store-operated Ca2+ entry (SOCE), which is mediated by STIM1 and Orai1 proteins. STIM1 was not localized to the ER-PM junction upon ER store depletion in RPGR, RPGRIP1 or RPGRIP1L KD cells. Our results demonstrate that the RPGR protein complex is required for regulating proteasomal activity and for modulating SOCE, which may contribute to the ciliopathy phenotype.

Ion channels in smooth muscle: regulation by the sarcoplasmic reticulum and mitochondria.

In smooth muscle, Ca(2+) regulates cell division, growth and cell death as well as providing the main trigger for contraction. Ion channels provide the major access route to elevate the cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) in smooth muscle by permitting Ca(2+) entry across the plasma membrane and release of the ion from intracellular Ca(2+) stores. The control of [Ca(2+)](c) relies on feedback modulation of the entry and release channels by Ca(2+) itself. Local rises in [Ca(2+)](c) may promote or inhibit channel activity directly or indirectly. The latter may arise from Ca(2+) regulation of ionic conductances in the plasma membrane to provide control of cell excitability and so [Ca(2+)](c) entry. Organelles such as mitochondria may also contribute significantly to the feedback regulation of ion channel activity by the control of Ca(2+) or redox status of the cell. This brief review describes the feedback regulation of Ca(2+) release from the internal Ca(2+) store and of plasma membrane excitability in smooth muscle.

Ca2+ microdomains in smooth muscle.

In smooth muscle, Ca(2+) controls diverse activities including cell division, contraction and cell death. Of particular significance in enabling Ca(2+) to perform these multiple functions is the cell's ability to localize Ca(2+) signals to certain regions by creating high local concentrations of Ca(2+) (microdomains), which differ from the cytoplasmic average. Microdomains arise from Ca(2+) influx across the plasma membrane or release from the sarcoplasmic reticulum (SR) Ca(2+) store. A single Ca(2+) channel can create a microdomain of several micromolar near (approximately 200 nm) the channel. This concentration declines quickly with peak rates of several thousand micromolar per second when influx ends. The high [Ca(2+)] and the rapid rates of decline target Ca(2+) signals to effectors in the microdomain with rapid kinetics and enable the selective activation of cellular processes. Several elements within the cell combine to enable microdomains to develop. These include the brief open time of ion channels, localization of Ca(2+) by buffering, the clustering of ion channels to certain regions of the cell and the presence of membrane barriers, which restrict the free diffusion of Ca(2+). In this review, the generation of microdomains arising from Ca(2+) influx across the plasma membrane and the release of the ion from the SR Ca(2+) store will be discussed and the contribution of mitochondria and the Golgi apparatus as well as endogenous modulators (e.g. cADPR and channel binding proteins) will be considered.

Flicker-assisted localization microscopy reveals altered mitochondrial architecture in hypertension.

Mitochondrial morphology is central to normal physiology and disease development. However, in many live cells and tissues, complex mitochondrial structures exist and morphology has been difficult to quantify. We have measured the shape of electrically-discrete mitochondria, imaging them individually to restore detail hidden in clusters and demarcate functional boundaries. Stochastic "flickers" of mitochondrial membrane potential were visualized with a rapidly-partitioning fluorophore and the pixel-by-pixel covariance of spatio-temporal fluorescence changes analyzed. This Flicker-assisted Localization Microscopy (FaLM) requires only an epifluorescence microscope and sensitive camera. In vascular myocytes, the apparent variation in mitochondrial size was partly explained by densely-packed small mitochondria. In normotensive animals, mitochondria were small spheres or rods. In hypertension, mitochondria were larger, occupied more of the cell volume and were more densely clustered. FaLM provides a convenient tool for increased discrimination of mitochondrial architecture and has revealed mitochondrial alterations that may contribute to hypertension.

Interactions between mitochondrial bioenergetics and cytoplasmic calcium in cultured cerebellar granule cells.

The mitochondrion has moved to the center stage in the drama of the life and death of the neuron. The mitochondrial membrane potential controls the ability of the organelle to generate ATP, generate reactive oxygen species and sequester Ca(2+) entering the cell. Each of these processes interact, and their deconvolution is far from trivial. The cultured cerebellar granule cell provides a model in which knowledge gained from studies on isolated mitochondria can be applied to study the role played by the organelles in the maintenance of Ca(2+) homeostasis in the cell under resting, stimulated and pathophysiological conditions. In particular, mitochondria play a complex role in the response of the neuron to excitotoxic stimulation of NMDA and AMPA-kainate selective glutamate receptors. One goal of research in this area is to provide clues as to possible ways in which modulators of mitochondrial function may be used as neuroprotective agents, since mitochondrial Ca(2+) accumulation seems to play a key role in glutamate excitotoxicity.