RESUMEN
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.
Asunto(s)
Mitocondrias/fisiología , Mitocondrias/ultraestructura , Músculo Liso Vascular/ultraestructura , Células Cultivadas , Dineínas/fisiología , Humanos , Cinesinas/fisiología , Microtúbulos/fisiología , Dinámicas Mitocondriales/fisiología , Membranas Mitocondriales/fisiología , Proteínas Mitocondriales/fisiología , Movimiento/fisiologíaRESUMEN
Mitochondrial Ca²âº uptake contributes important feedback controls to limit the time course of Ca²âº signals. Mitochondria regulate cytosolic [Ca²âº] over an exceptional breath of concentrations (~200 nM to >10 µM) to provide a wide dynamic range in the control of Ca²âº signals. Ca²âº 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 Ca²âº 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 Ca²âº signaling in native smooth muscle.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , Potencial de la Membrana Mitocondrial/fisiología , Mitocondrias Musculares/metabolismo , Músculo Liso Vascular/metabolismo , Animales , Humanos , Fuerza Protón-Motriz/fisiologíaRESUMEN
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.
Asunto(s)
Calcio/metabolismo , Compuestos de Anilina/metabolismo , Animales , Cobayas , Técnicas In Vitro , Masculino , Microscopía Fluorescente , Miocitos del Músculo Liso/metabolismo , Técnicas de Placa-Clamp , Xantenos/metabolismoRESUMEN
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 (â¼10%) 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.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Microdominios de Membrana/metabolismo , Miocitos del Músculo Liso/metabolismo , Receptor Muscarínico M3/metabolismo , Animales , Señalización del Calcio/efectos de los fármacos , Carbacol/farmacología , Colon/efectos de los fármacos , Colon/metabolismo , Cobayas , Masculino , Microdominios de Membrana/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Fotólisis/efectos de los fármacos , Isoformas de Proteínas/metabolismo , Transporte de Proteínas/efectos de los fármacosRESUMEN
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.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Membrana Celular/metabolismo , Animales , Calcio/química , Citoplasma/metabolismo , Humanos , Cinética , Microscopía Fluorescente/métodos , Modelos Biológicos , Transducción de SeñalRESUMEN
The cytosolic Ca²âº concentration ([Ca²âº]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 [Ca²âº]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 Ca²âº. However, mitochondria's reported low affinity for Ca²âº is thought to require the organelle to be positioned close to ion channels and within a microdomain of high [Ca²âº]. 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 Ca²âº signals and IP3 receptor (IP3R) cluster activity. These observations suggest mitochondria are positioned within the high [Ca²âº] 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 Ca²âº 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 Ca²âº to enable uptake to occur over the normal physiological Ca²âº range (<1 µM). Thus, in smooth muscle, mitochondria regulate Ca²âº signals exerting effects over a large range of [Ca²âº] (â¼200 nM to at least tens of micromolar) to provide a wide dynamic range in the control of Ca²âº signals.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Citosol/metabolismo , Mitocondrias/metabolismo , Miocitos del Músculo Liso/metabolismo , Animales , Canales de Calcio/metabolismo , Humanos , Potencial de la Membrana Mitocondrial , Miocitos del Músculo Liso/citologíaRESUMEN
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.
Asunto(s)
Calcio/metabolismo , Mitocondrias/fisiología , Animales , Señalización del Calcio , Proteínas de Transporte de Catión/metabolismo , Retículo Endoplásmico/metabolismo , Humanos , Potencial de la Membrana Mitocondrial , Mitocondrias/metabolismoRESUMEN
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.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Calcio/metabolismo , Carbacol/farmacología , Inositol 1,4,5-Trifosfato/metabolismo , Animales , Compuestos de Diazonio/metabolismo , Cobayas , Masculino , Potenciales de la Membrana/efectos de los fármacos , Fenoxiacetatos/metabolismo , Fotólisis/efectos de los fármacosRESUMEN
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.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Mitocondrias/metabolismo , Miocitos del Músculo Liso/metabolismo , Animales , Señalización del Calcio/efectos de los fármacos , Cobayas , Inositol 1,4,5-Trifosfato/farmacología , Masculino , Miocitos del Músculo Liso/efectos de los fármacosRESUMEN
In smooth muscle, the gating of dihydropyridine-sensitive Ca(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(2+) influx and determining the bulk average cytoplasmic Ca(2+) concentration. Here, the contribution of voltage-dependent and constitutively active channel behavior to Ca(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(2+) current (I(Ca)) and evoked a rise in [Ca(2+)] in each of the subplasma membrane space and bulk cytoplasm. In various regions of the bulk cytoplasm the [Ca(2+)] increase ([Ca(2+)](c)) was approximately uniform, whereas that of the subplasma membrane space ([Ca(2+)](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(2+) channels (clusters) across the sarcolemma, and their activation appeared consistent with normal voltage-dependent behavior. Indeed, in the present study, dihydropyridine-sensitive Ca(2+) channels were not normally constitutively active. The repetitive localized [Ca(2+)](PM) rises ("persistent Ca(2+) sparklets") that characterize constitutively active channels were observed rarely (2 of 306 cells). Neither did dihydropyridine-sensitive constitutively active Ca(2+) channels regulate the bulk average [Ca(2+)](c). A dihydropyridine blocker of Ca(2+) channels, nimodipine, which blocked I(Ca) and accompanying [Ca(2+)](c) rise, reduced neither the resting bulk average [Ca(2+)](c) (at -70 mV) nor the rise in [Ca(2+)](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(2+) channels appear clustered in certain regions of the plasma membrane, constitutive activity is unlikely to play a major role in [Ca(2+)](c) regulation. The stochastic, voltage-dependent activity of the channel provides the major mechanism to generate rises in [Ca(2+)].
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Endotelio Vascular/metabolismo , Tracto Gastrointestinal/metabolismo , Líquido Intracelular/metabolismo , Músculo Liso Vascular/metabolismo , Potenciales de Acción/fisiología , Animales , Cobayas , MasculinoRESUMEN
Whether or not the sarcoplasmic reticulum (SR) is a continuous, interconnected network surrounding a single lumen or comprises multiple, separate Ca2+ pools was investigated in voltage-clamped single smooth muscle cells using local photolysis of caged compounds and Ca2+ imaging. The entire SR could be depleted or refilled from one small site via either inositol 1,4,5-trisphosphate receptors (IP3R) or ryanodine receptors (RyR) suggesting the SR is luminally continuous and that Ca2+ may diffuse freely throughout. Notwithstanding, regulation of the opening of RyR and IP3R, by the [Ca2+] within the SR, may create several apparent SR elements with various receptor arrangements. IP3R and RyR may appear to exist entirely on a single store, and there may seem to be additional SR elements that express either only RyR or only IP3R. The various SR receptor arrangements and apparently separate Ca2+ storage elements exist in a single luminally continuous SR entity.
Asunto(s)
Calcio/metabolismo , Músculo Liso/metabolismo , Retículo Sarcoplasmático/metabolismo , Animales , Sitios de Unión , Compuestos de Boro/farmacología , Cafeína/farmacología , Electrofisiología , Cobayas , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Iones , Masculino , Modelos Biológicos , Fotólisis , Canal Liberador de Calcio Receptor de Rianodina/metabolismoRESUMEN
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.
Asunto(s)
Señalización del Calcio , Canales Iónicos/metabolismo , Mitocondrias/metabolismo , Músculo Liso/metabolismo , Retículo Sarcoplasmático/metabolismo , Animales , Calcio/metabolismo , Membrana Celular/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismoRESUMEN
Smooth muscle is activated by plasma-membrane-acting agonists that induce inositol (1,4,5)-trisphosphate [Ins(1,4,5)P(3)] to release Ca(2+) from the intracellular sarcoplasmic reticulum (SR) Ca(2+) store. Increased concentrations of agonist evoke a concentration-dependent graded release of Ca(2+) in a process called 'quantal' Ca(2+) release. Such a graded release seems to be incompatible with both the finite capacity of the SR store and the positive-feedback Ca(2+)-induced Ca(2+) release (CICR)-like process that is operative at Ins(1,4,5)P(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(3), or Ins(1,4,5)P(3) receptor opening being controlled by the Ca(2+) concentration within the SR. Here, we suggest that the regulation of Ins(1,4,5)P(3) receptors by the Ca(2+) concentration within the SR explains the quantal Ca(2+)-release process and the apparent existence of multiple Ca(2+) stores in smooth muscle.
Asunto(s)
Calcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/fisiología , Músculo Liso/metabolismo , Animales , Citoplasma/metabolismo , Humanos , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Retículo Sarcoplasmático/metabolismoRESUMEN
The effects of the phytoestrogens phloretin and phloridzin on Ca(2+) handling, cell shortening, the action potential, and Ca(2+) and K(+) currents in freshly isolated cardiac myocytes from rat ventricle were examined. Phloretin increased the amplitude and area and decreased the rate of decline of electrically evoked Ca(2+) transients in the myocytes. These effects were accompanied by an increase in the Ca(2+) load of the sarcoplasmic reticulum, as determined by the area of caffeine-evoked Ca(2+) transients. An increase in the extent of shortening of the myocytes in response to electrically evoked action potentials was also observed in the presence of phloretin. To further examine possible mechanisms contributing to the observed changes in Ca(2+) handling and contractility, the effects of phloretin on the cardiac action potential and plasma membrane Ca(2+) and K(+) currents were examined. Phloretin markedly increased the action potential duration in the myocytes, and it inhibited the Ca(2+)-independent transient outward K(+) current (I(to)). The inwardly rectifying K(+) current, the sustained outward delayed rectifier K(+) current, and L-type Ca(2+) currents were not significantly different in the presence and absence of phloretin, nor was there any evidence that the Na(+)/Ca(2+) exchanger was affected. The effects of phloretin on Ca(2+) handling in the myocytes are consistent with its effects on I(to). Phloridzin did not significantly alter the amplitude or area of electrically evoked Ca(2+) transients in the myocytes, nor did it have detectable effects on the sarcoplasmic reticulum Ca(2+) load, cell shortening, or the action potential.
Asunto(s)
Potenciales de Acción/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Canales Iónicos/fisiología , Miocitos Cardíacos/efectos de los fármacos , Floretina/farmacología , Florizina/farmacología , Animales , Cafeína/farmacología , Calcio/metabolismo , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo L/fisiología , Forma de la Célula/efectos de los fármacos , Citoplasma/metabolismo , Estimulación Eléctrica , Electrofisiología , Canales Iónicos/metabolismo , Masculino , Miocitos Cardíacos/citología , Miocitos Cardíacos/fisiología , Canales de Potasio/metabolismo , Canales de Potasio/fisiología , Ratas , Ratas Sprague-Dawley , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/metabolismoRESUMEN
Phytoestrogens are naturally occurring estrogenic compounds found in plants and plant products. These compounds are also known to exert cellular effects independent of their interactions with estrogen receptors. We studied the effects of the phytoestrogens phloretin, phloridzin, genistein, and biochanin A on Ca(2+) uptake into the cardiac muscle sarcoplasmic reticulum (SR). Genistein and biochanin A did not affect SR Ca(2+) uptake. On the other hand, phloretin and phloridzin decreased the maximum velocity of SR Ca(2+) uptake but did not affect the Hill coefficient or the Ca(2+) sensitivity of uptake. Measurements of the ATPase activity of the cardiac SR Ca(2+) pump (SERCA2a) revealed direct inhibitory effects of phloretin and phloridzin on SERCA2a. Neither compound induced a detectable change in the permeability of the SR membrane to Ca(2+). These results indicate that phloretin and phloridzin inhibit cardiac SR Ca(2+) uptake by directly inhibiting SERCA2a.