A mechanistic systems pharmacology modeling platform to investigate the effect of PD-L1 expression heterogeneity and dynamics on the efficacy of PD-1 and PD-L1 blocking antibodies in cancer.

Tumors have developed multitude of ways to evade immune response and suppress cytotoxic T cells. Programed cell death protein 1 (PD-1) and programed cell death ligand 1 (PD-L1) are immune checkpoints that when activated, rapidly inactivate the cytolytic activity of T cells. Expression heterogeneity of PD-L1 and the surface receptor dynamics of both PD-1 and PD-L1 may be important parameters in modulating the immune response. PD-L1 is expressed on both tumor and non-tumor immune cells and this differential expression reflects different aspects of anti-tumor immunity. Here, we developed a mechanistic computational model to investigate the role of PD-1 and PD-L1 dynamics in modulating the efficacy of PD-1 and PD-L1 blocking antibodies. Our model incorporates immunological synapse restricted interaction of PD-1 and PD-L1, basal parameters for receptor dynamics, and T cell interaction with tumor and non-tumor immune cells. Simulations predict the existence of a threshold in PD-1 expression above which there is no efficacy for both anti-PD-1 and anti-PD-L1. Model also predicts that anti-tumor response is more sensitive to PD-L1 expression on non-tumor immune cells than tumor cells. New combination strategies are suggested that may enhance efficacy in resistant cases such as combining anti-PD-1 with a low dose of anti-PD-L1 or with inhibitors of PD-L1 recycling and synthesis. Another combination strategy suggested by the model is the combination of anti-PD-1 and anti-PD-L1 with enhancers of PD-L1 degradation rate. Virtual patients are then generated to test specific biomarkers of response. Intriguing predictions that emerge from the virtual patient simulations are that PD-1 blocking antibody results in higher response rate than PD-L1 blockade and that PD-L1 expression density on non-tumor immune cells rather than tumor cells is a predictor of response.

Computational modeling of synergistic interaction between αVß3 integrin and VEGFR2 in endothelial cells: Implications for the mechanism of action of angiogenesis-modulating integrin-binding peptides.

Cooperation between VEGFR2 and integrin αVß3 is critical for neovascularization in wound healing, cardiovascular ischemic diseases, ocular diseases, and tumor angiogenesis. In the present study, we developed a rule-based computational model to investigate the potential mechanism by which the Src-induced integrin association with VEGFR2 enhances VEGFR2 activation. Simulations demonstrated that the main function of integrin is to reduce the degradation of VEGFR2 and hence stabilize the activation signal. In addition, receptor synthesis rate and recruitment from internal compartment were found to be sensitive determinants of the activation state of VEGFR2. The model was then applied to simulate the effect of integrin-binding peptides such as tumstatin and cilengitide on VEGFR2 signaling. Further, computational modeling proposed potential molecular mechanisms for the angiogenesis-modulating activity of other integrin-binding peptides. The model highlights the complexity of the crosstalk between αVß3 integrin and VEGFR2 and the necessity of utilizing models to elucidate potential mechanisms in angiogenesis-modulating peptide therapy.

Computational investigation of sphingosine kinase 1 (SphK1) and calcium dependent ERK1/2 activation downstream of VEGFR2 in endothelial cells.

Vascular endothelial growth factor (VEGF) is a powerful regulator of neovascularization. VEGF binding to its cognate receptor, VEGFR2, activates a number of signaling pathways including ERK1/2. Activation of ERK1/2 is experimentally shown to involve sphingosine kinase 1 (SphK1) activation and its calcium-dependent translocation downstream of ERK1/2. Here we construct a rule-based computational model of signaling downstream of VEGFR2, by including SphK1 and calcium positive feedback mechanisms, and investigate their consequences on ERK1/2 activation. The model predicts the existence of VEGF threshold in ERK1/2 activation that can be continuously tuned by cellular concentrations of SphK1 and sphingosine 1 phosphate (S1P). The computer model also predicts powerful effects of perturbations in plasma and ER calcium pump rates and the current through the CRAC channels on ERK1/2 activation dynamics, highlighting the critical role of intracellular calcium in shaping the pERK1/2 signal. The model is then utilized to simulate anti-angiogenic therapeutic interventions targeting VEGFR2-ERK1/2 axis. Simulations indicate that monotherapies that exclusively target VEGFR2 phosphorylation, VEGF, or VEGFR2 are ineffective in shutting down signaling to ERK1/2. By simulating therapeutic strategies that target multiple nodes of the pathway such as Raf and SphK1, we conclude that combination therapy should be much more effective in blocking VEGF signaling to EKR1/2. The model has important implications for interventions that target signaling pathways in angiogenesis relevant to cancer, vascular diseases, and wound healing.

Novel fluorescence resonance energy transfer-based reporter reveals differential calcineurin activation in neonatal and adult cardiomyocytes.

Novel fluorescence resonance energy transfer-based genetically encoded reporters of calcineurin are constructed by fusing the two subunits of calcineurin with P2A-based linkers retaining the expected native conformation of calcineurin. Calcineurin reporters display robust responses to calcium transients in HEK293 cells. The sensor responses are correlated with NFATc1 translocation dynamics in HEK293 cells. The sensors are uniformly distributed in neonatal myocytes and respond efficiently to single electrically evoked calcium transients and show cumulative activation at frequencies of 0.5 and 1 Hz. In adult myocytes, the calcineurin sensors appear to be localized to the cardiac z-lines, and respond to cumulative calcium transients at frequencies of 0.5 and 1 Hz. The phosphatase calcineurin is a central component of many calcium signalling pathways, relaying calcium signals from the plasma membrane to the nucleus. It has critical functions in a multitude of systems, including immune, cardiac and neuronal. Given the widespread importance of calcineurin in both normal and pathological conditions, new tools that elucidate the spatiotemporal dynamics of calcineurin activity would be invaluable. Here we develop two separate genetically encoded fluorescence resonance energy transfer (FRET)-based sensors of calcineurin activation, DuoCaN and UniCaN. Both sensors showcase a large dynamic range and rapid response kinetics, differing primarily in the linker structure between the FRET pairs. Both sensors were calibrated in HEK293 cells and their responses correlated well with NFAT translocation to the nucleus, validating the biological relevance of the sensor readout. The sensors were subsequently expressed in neonatal rat ventricular myocytes and acutely isolated adult guinea pig ventricular myocytes. Both sensors demonstrated robust responses in myocytes and revealed kinetic differences in calcineurin activation during changes in pacing rate for neonatal versus adult myocytes. Finally, mathematical modelling combined with quantitative FRET measurements provided novel insights into the kinetics and integration of calcineurin activation in response to myocyte Ca transients. In all, DuoCaN and UniCaN stand as valuable new tools for understanding the role of calcineurin in normal and pathological signalling.

Eliciting the antitumor immune response with a conditionally activated PD-L1 targeting antibody analyzed with a quantitative systems pharmacology model.

Conditionally activated molecules, such as Probody therapeutics (PbTx), have recently been investigated to improve antitumoral response while reducing systemic toxicity. PbTx are engineered to be proteolytically activated by proteases that are preferentially active locally in the tumor microenvironment (TME). Here, we perform an exploratory study using our recently published quantitative systems pharmacology model, previously validated for other drugs, to evaluate the effectiveness and targeting specificity of an anti-PD-L1 PbTx compared to the non-modified antibody. We have informed the model using the PbTx dynamics and pharmacokinetics published in the literature for anti-PD-L1 in patients with triple-negative breast cancer (TNBC). Our results suggest masking of the antibody slightly decreases its efficacy, while increasing the localization of active therapeutic component in the TME. We also perform a parameter optimization for the PbTx design and drug dosing regimens to maximize the response rate. Although our results are specific to the case of TNBC, our findings are generalizable to any conditionally activated PbTx molecule in solid tumors and suggest that design of a highly effective and selective PbTx is feasible.

Combining Multikinase Tyrosine Kinase Inhibitors Targeting the Vascular Endothelial Growth Factor and Cluster of Differentiation 47 Signaling Pathways Is Predicted to Increase the Efficacy of Antiangiogenic Combination Therapies.

Angiogenesis is a critical step in tumor growth, development, and invasion. Nascent tumor cells secrete vascular endothelial growth factor (VEGF) that significantly remodels the tumor microenvironment through interaction with multiple receptors on vascular endothelial cells, including type 2 VEGF receptor (VEGFR2). The complex pathways initiated by VEGF binding to VEGFR2 lead to enhanced proliferation, survival, and motility of vascular endothelial cells and formation of a new vascular network, enabling tumor growth. Antiangiogenic therapies that inhibit VEGF signaling pathways were among the first drugs that targeted stroma rather than tumor cells. Despite improvements in progression-free survival and higher response rates relative to chemotherapy in some types of solid tumors, the impact on overall survival (OS) has been limited, with the majority of tumors eventually relapsing due to resistance or activation of alternate angiogenic pathways. Here, we developed a molecularly detailed computational model of endothelial cell signaling and angiogenesis-driven tumor growth to investigate combination therapies targeting different nodes of the endothelial VEGF/VEGFR2 signaling pathway. Simulations predicted a strong threshold-like behavior in extracellular signal-regulated kinases 1/2 (ERK1/2) activation relative to phosphorylated VEGFR2 levels, as continuous inhibition of at least 95% of receptors was necessary to abrogate phosphorylated ERK1/2 (pERK1/2). Combinations with mitogen-activated protein kinase/ERK kinase (MEK) and spingosine-1-phosphate inhibitors were found to be effective in overcoming the ERK1/2 activation threshold and abolishing activation of the pathway. Modeling results also identified a mechanism of resistance whereby tumor cells could reduce pERK1/2 sensitivity to inhibitors of VEGFR2 by upregulation of Raf, MEK, and sphingosine kinase 1 (SphK1), thus highlighting the need for deeper investigation of the dynamics of the crosstalk between VEGFR2 and SphK1 pathways. Inhibition of VEGFR2 phosphorylation was found to be more effective at blocking protein kinase B, also known as AKT, activation; however, to effectively abolish AKT activation, simulations identified Axl autophosphorylation or the Src kinase domain as potent targets. Simulations also supported activating cluster of differentiation 47 (CD47) on endothelial cells as an effective combination partner with tyrosine kinase inhibitors to inhibit angiogenesis signaling and tumor growth. Virtual patient simulations supported the effectiveness of CD47 agonism in combination with inhibitors of VEGFR2 and SphK1 pathways. Overall, the rule-based system model developed here provides new insights, generates novel hypothesis, and makes predictions regarding combinations that may enhance the OS with currently approved antiangiogenic therapies.

TAK-676: A Novel Stimulator of Interferon Genes (STING) Agonist Promoting Durable IFN-dependent Antitumor Immunity in Preclinical Studies.

Oncology therapies targeting the immune system have improved patient outcomes across a wide range of tumor types, but resistance due to an inadequate T-cell response in a suppressive tumor microenvironment (TME) remains a significant problem. New therapies that activate an innate immune response and relieve this suppression may be beneficial to overcome this hurdle. TAK-676 is a synthetic novel stimulator of interferon genes (STING) agonist designed for intravenous administration. Here we demonstrate that TAK-676 dose-dependently triggers activation of the STING signaling pathway and activation of type I interferons. Furthermore, we show that TAK-676 is a highly potent modulator of both the innate and adaptive immune system and that it promotes the activation of dendritic cells, natural killer cells, and T cells in preclinical models. In syngeneic murine tumor models in vivo, TAK-676 induces dose-dependent cytokine responses and increases the activation and proliferation of immune cells within the TME and tumor-associated lymphoid tissue. We also demonstrate that TAK-676 dosing results in significant STING-dependent antitumor activity, including complete regressions and durable memory T-cell immunity. We show that TAK-676 is well tolerated, exhibits dose-proportional pharmacokinetics in plasma, and exhibits higher exposure in tumor. The intravenous administration of TAK-676 provides potential treatment benefit in a broad range of tumor types. Further study of TAK-676 in first-in-human phase I trials is ongoing. Significance: TAK-676 is a novel systemic STING agonist demonstrating robust activation of innate and adaptive immune activity resulting in durable antitumor responses within multiple syngeneic tumor models. Clinical investigation of TAK-676 is ongoing.

Computer Simulation of TSP1 Inhibition of VEGF-Akt-eNOS: An Angiogenesis Triple Threat.

The matricellular protein thrombospondin-1 (TSP1) is a potent inhibitor of angiogenesis. Specifically, TSP1 has been experimentally shown to inhibit signaling downstream of vascular endothelial growth factor (VEGF). The molecular mechanism of this inhibition is not entirely clear. We developed a detailed computational model of VEGF signaling to Akt-endothelial nitric oxide synthase (eNOS) to investigate the quantitative molecular mechanism of TSP1 inhibition. The model demonstrated that TSP1 acceleration of VEGFR2 degradation is sufficient to explain the inhibition of VEGFR2 and eNOS phosphorylation. However, Akt inhibition requires TSP1-induced phosphatase recruitment to VEGFR2. The model was then utilized to test various strategies for the rescue of VEGF signaling to Akt and eNOS. Inhibiting TSP1 was predicted to be not as effective as CD47 depletion in rescuing signaling to Akt. The model further predicts that combination strategy involving depletion of CD47 and inhibition of TSP1 binding to CD47 is necessary for effective recovery of signaling to eNOS. In all, computational modeling offers insight to molecular mechanisms involving TSP1 interaction with VEGF signaling and provides strategies for rescuing angiogenesis by targeting TSP1-CD47 axis.

Three-Dimensional Transport Model for Intravitreal and Suprachoroidal Drug Injection.

Purpose: Quantitative understanding of the transport of therapeutic macromolecules following intraocular injections is critical for the design of efficient strategies in treating eye diseases, such as neovascular (wet) age-related macular degeneration (AMD) and macular edema (ME). Antiangiogenic treatments, such as neutralizing antibodies against VEGF or recently characterized antiangiogenic peptides, have shown promise in slowing disease progression. Methods: We developed a comprehensive three-dimensional (3D) transport model for intraocular injections using published data on drug distribution in rabbit eyes following intravitreal and suprachoroidal (SC) injection of sodium fluorescein (SF), bevacizumab, and ranibizumab. The model then was applied to evaluate the distribution of small molecules and antiangiogenic proteins following intravitreal and SC injections in human eyes. Results: The model predicts that intravitreally administered molecules are substantially mixed within the vitreous following injection, and that the long-term behavior of the injected drug does not depend on the initial mixing. Ocular pharmacokinetics of different drugs is sensitive to different clearance mechanisms. Effective retinal drug delivery is impacted by RPE permeability. For VEGF antibody, intravitreal injection provides sustained delivery to the retina, whereas SC injection provides more efficient, but short-lived, retinal delivery for smaller-sized molecules. Long-term suppression of neovascularization through SC administration of antiangiogenic drugs necessitates frequent injection or sustained delivery, such as microparticle-based delivery of antiangiogenic peptides. Conclusions: A comprehensive 3D model for intravitreal and SC drug injection is developed to provide a framework and platform for testing drug delivery routes and sustained delivery devices for new and existing drugs.

Biomimetic peptide display from a polymeric nanoparticle surface for targeting and antitumor activity to human triple-negative breast cancer cells.

While poly(lactic-co-glycolic acid)-block-polyethylene glycol (PLGA-PEG) nanoparticles (NPs) can encapsulate drug cargos and prolong circulation times, they show nonspecific accumulation in off-target tissues. Targeted delivery of drugs to tumor tissue and tumor vasculature is a promising approach for treating solid tumors while enhancing specificity and reducing systemic toxicity. AXT050, a collagen-IV derived peptide with both antitumor and antiangiogenic properties, is shown to bind to tumor-associated integrins with high affinity, which leads to targeted accumulation in tumor tissue. AXT050 conjugated to PLGA-PEG NPs at precisely controlled surface density functions both as a targeting agent to human tumor cells and demonstrates potential for simultaneous antitumorigenic and antiangiogenic activity. These targeted NPs cause inhibition of adhesion and proliferation in vitro when added to human triple-negative breast cancer cells and microvascular endothelial cells through binding to integrin αV ß3 . Furthermore, we find an in vivo biphasic relationship between tumor targeting and surface coating density of NPs coated with AXT050. NPs with an intermediate level of 10% peptide surface coating show approximately twofold greater accumulation in tumors and lower accumulation in the liver compared to nontargeted PLGA-PEG NPs in a murine biodistribution model. Display of biomimetic peptides from NP surfaces to both target and inhibit cancer cells has the potential to enhance the activity of cancer nanomedicines. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1753-1764, 2018.

Inhibition of VEGFR2 Activation and Its Downstream Signaling to ERK1/2 and Calcium by Thrombospondin-1 (TSP1): In silico Investigation.

VEGF signaling through VEGFR2 is a central regulator of the angiogenic response. Inhibition of VEGF signaling by the stress-induced matricellular protein TSP1 plays a role in modulating the angiogenic response to VEGF in both health and disease. TSP1 binding to CD47 inhibits VEGFR2 activation. The full implications of this inhibitory interaction are unknown. We developed a detailed rule-based computational model to inquire if TSP1-CD47 signaling through VEGF had downstream effects upon ERK1/2 and calcium. Our Simulations suggest that enhanced degradation of VEGFR2 initiated by the binding of TSP1 to CD47 is sufficient to explain the inhibition of VEGFR2 phosphorylation, calcium elevation, and ERK1/2 activation downstream of VEGF. A complementary mechanism involving the recruitment of phosphatases to the VEGFR2 complex with consequent increase in the rate of receptor dephosphorylation may augment the inhibition of the VEGF signal. The model was then utilized to simulate the effect of inhibiting external TSP1 or the depletion of CD47 as potential therapeutic strategies in restoring VEGF signaling. Results suggest that depleting CD47 is a more efficient strategy in inhibiting the effects of TSP1/CD47 on VEGF signaling. Our results highlight the utility of in silico investigations in elucidating and clarifying molecular mechanisms at the intersection of TSP1 and VEGF biology and in differentiating between competing pro-angiogenic therapeutic strategies relevant to peripheral arterial disease (PAD) and wound healing.

Corrigendum: Inhibition of VEGFR2 Activation and Its Downstream Signaling to ERK1/2 and Calcium by Thrombospondin-1 (TSP1): In silico Investigation.

[This corrects the article on p. 48 in vol. 8, PMID: 28220078.].

Towards a Unified Theory of Calmodulin Regulation (Calmodulation) of Voltage-Gated Calcium and Sodium Channels.

Voltage-gated Na and Ca(2+) channels represent two major ion channel families that enable myriad biological functions including the generation of action potentials and the coupling of electrical and chemical signaling in cells. Calmodulin regulation (calmodulation) of these ion channels comprises a vital feedback mechanism with distinct physiological implications. Though long-sought, a shared understanding of the channel families remained elusive for two decades as the functional manifestations and the structural underpinnings of this modulation often appeared to diverge. Here, we review recent advancements in the understanding of calmodulation of Ca(2+) and Na channels that suggest a remarkable similarity in their regulatory scheme. This interrelation between the two channel families now paves the way towards a unified mechanistic framework to understand vital calmodulin-dependent feedback and offers shared principles to approach related channelopathic diseases. An exciting era of synergistic study now looms.

Continuously tunable Ca(2+) regulation of RNA-edited CaV1.3 channels.

CaV1.3 ion channels are dominant Ca(2+) portals into pacemaking neurons, residing at the epicenter of brain rhythmicity and neurodegeneration. Negative Ca(2+) feedback regulation of CaV1.3 channels (CDI) is therefore critical for Ca(2+) homeostasis. Intriguingly, nearly half the CaV1.3 transcripts in the brain are RNA edited to reduce CDI and influence oscillatory activity. It is then mechanistically remarkable that this editing occurs precisely within an IQ domain, whose interaction with Ca(2+)-bound calmodulin (Ca(2+)/CaM) is believed to induce CDI. Here, we sought the mechanism underlying the altered CDI of edited channels. Unexpectedly, editing failed to attenuate Ca(2+)/CaM binding. Instead, editing weakened the prebinding of Ca(2+)-free CaM (apoCaM) to channels, which proves essential for CDI. Thus, editing might render CDI continuously tunable by fluctuations in ambient CaM, a prominent effect we substantiate in substantia nigral neurons. This adjustability of Ca(2+) regulation by CaM now looms as a key element of CNS Ca(2+) homeostasis.

Dynamic switching of calmodulin interactions underlies Ca2+ regulation of CaV1.3 channels.

Calmodulin regulation of CaV channels is a prominent Ca(2+) feedback mechanism orchestrating vital adjustments of Ca(2+) entry. The long-held structural correlation of this regulation has been Ca(2+)-bound calmodulin, complexed alone with an IQ domain on the channel carboxy terminus. Here, however, systematic alanine mutagenesis of the entire carboxyl tail of an L-type CaV1.3 channel casts doubt on this paradigm. To identify the actual molecular states underlying channel regulation, we develop a structure-function approach relating the strength of regulation to the affinity of underlying calmodulin/channel interactions, by a Langmuir relation (individually transformed Langmuir analysis). Accordingly, we uncover frank exchange of Ca(2+)-calmodulin to interfaces beyond the IQ domain, initiating substantial rearrangements of the calmodulin/channel complex. The N-lobe of Ca(2+)-calmodulin binds an N-terminal spatial Ca(2+) transforming element module on the channel amino terminus, whereas the C-lobe binds an EF-hand region upstream of the IQ domain. This system of structural plasticity furnishes a next-generation blueprint for CaV channel modulation.

A mathematical model of electrotonic interactions between ventricular myocytes and fibroblasts.

Functional intercellular coupling has been demonstrated among networks of cardiac fibroblasts, as well as between fibroblasts and atrial or ventricular myocytes. In this study, the consequences of these interactions were examined by implementing the ten Tusscher model of the human ventricular action potential, and coupling it to our electrophysiological models for mammalian ventricular fibroblasts. Our simulations reveal significant electrophysiological consequences of coupling between 1 and 4 fibroblasts to a single ventricular myocyte. These include alterations in plateau height and/or action potential duration (APD) and changes in underlying ionic currents. Two series of simulations were carried out. First, fibroblasts were modeled as a spherical cell with a capacitance of 6.3 pF and an ohmic membrane resistance of 10.7 G Omega. When these "passive" fibroblasts were coupled to a myocyte, they caused slight prolongation of APD with no changes in the plateau, threshold for firing, or rate of initial depolarization. In contrast, when the same myocyte-fibroblast complexes were modeled after addition of the time- and voltage-gated K(+) currents that are expressed in fibroblasts, much more pronounced effects were observed: the plateau height of the action potential was reduced and the APD shortened significantly. In addition, each fibroblast exhibited significant electrotonic depolarizations in response to each myocyte action potential and the resting potential of the fibroblasts closely approximated the resting potential of the coupled ventricular myocyte.

Inhibition of cAMP-dependent protein kinase under conditions occurring in the cardiac dyad during a Ca2+ transient.

The space between the t-tubule invagination and the sarcoplasmic reticulum (SR) membrane, the dyad, in ventricular myocytes has been predicted to experience very high [Ca2+] for short periods of time during a Ca2+ transient. The dyadic space accommodates many protein kinases responsible for the regulation of Ca2+ handling proteins of the cell. We show in vitro that cAMP-dependent protein kinase (PKA) is inhibited by high [Ca2+] through a shift in the ratio of CaATP/MgATP toward CaATP. We further generate a three-dimensional mathematical model of Ca2+ and ATP diffusion within dyad. We use this model to predict the extent to which PKA would be inhibited by an increased CaATP/MgATP ratio during a Ca2+ transient in the dyad in vivo. Our results suggest that under normal physiological conditions a myocyte paced at 1 Hz would experience up to 55% inhibition of PKA within the cardiac dyad, with inhibition averaging 5% throughout the transient, an effect which becomes more pronounced as the myocyte contractile frequency increases (at 7 Hz, PKA inhibition averages 28% across the dyad throughout the duration of a Ca2+ transient).

Ca2+ regulation in the near-membrane microenvironment in smooth muscle cells.

The microenvironment between the plasma membrane and the near-membrane sarcoplasmic reticulum (SR) may play an important role in Ca(2+) regulation in smooth muscle cells. We used a three-dimensional mathematical model of Ca(2+) diffusion and regulation and experimental measurements of SR Ca(2+) uptake and the distribution of the SR in isolated smooth muscle cells to predict the extent that the near-membrane SR could load Ca(2+) after the opening of single plasma membrane Ca(2+) channels. We also modeled the effect of SR uptake on 1), single-channel Ca(2+) transients in the near-membrane space; 2), the association of Ca(2+) with Ca(2+) buffers in this space; and 3), the amount of Ca(2+) reaching the central cytoplasm of the cell. Our results indicate that, although single-channel Ca(2+) transients could increase SR Ca(2+) to a certain extent, SR Ca(2+) uptake is not rapid enough to greatly affect the magnitude of these transients or their spread to the central cytoplasm unless the Ca(2+) uptake rate of the peripheral SR is an order-of-magnitude higher than the mean rate derived from our experiments. Immunofluorescence imaging, however, did not reveal obvious differences in the density of SR Ca(2+) pumps or phospholamban between the peripheral and central SR in smooth muscle cells.