Wnt signaling regulates acetylcholine receptor translocation and synaptic plasticity in the adult nervous system.

The adult nervous system is plastic, allowing us to learn, remember, and forget. Experience-dependent plasticity occurs at synapses--the specialized points of contact between neurons where signaling occurs. However, the mechanisms that regulate the strength of synaptic signaling are not well understood. Here, we define a Wnt-signaling pathway that modifies synaptic strength in the adult nervous system by regulating the translocation of one class of acetylcholine receptors (AChRs) to synapses. In Caenorhabditis elegans, we show that mutations in CWN-2 (Wnt ligand), LIN-17 (Frizzled), CAM-1 (Ror receptor tyrosine kinase), or the downstream effector DSH-1 (disheveled) result in similar subsynaptic accumulations of ACR-16/α7 AChRs, a consequent reduction in synaptic current, and predictable behavioral defects. Photoconversion experiments revealed defective translocation of ACR-16/α7 to synapses in Wnt-signaling mutants. Using optogenetic nerve stimulation, we demonstrate activity-dependent synaptic plasticity and its dependence on ACR-16/α7 translocation mediated by Wnt signaling via LIN-17/CAM-1 heteromeric receptors.

Gene-vegetarianism interactions in calcium, estimated glomerular filtration rate, and testosterone identified in genome-wide analysis across 30 biomarkers.

We examined the associations of vegetarianism with metabolic biomarkers using traditional and genetic epidemiology. First, we addressed inconsistencies in self-reported vegetarianism among UK Biobank participants by utilizing data from two dietary surveys to find a cohort of strict European vegetarians (N = 2,312). Vegetarians were matched 1:4 with nonvegetarians for non-genetic association analyses, revealing significant effects of vegetarianism in 15 of 30 biomarkers. Cholesterol measures plus vitamin D were significantly lower in vegetarians, while triglycerides were higher. A genome-wide association study revealed no genome-wide significant (GWS; 5×10-8) associations with vegetarian behavior. We performed genome-wide gene-vegetarianism interaction analyses for the biomarkers, and detected a GWS interaction impacting calcium at rs72952628 (P = 4.47×10-8). rs72952628 is in MMAA, a B12 metabolic pathway gene; B12 has major deficiency potential in vegetarians. Gene-based interaction tests revealed two significant genes, RNF168 in testosterone (P = 1.45×10-6) and DOCK4 in estimated glomerular filtration rate (eGFR) (P = 6.76×10-7), which have previously been associated with testicular and renal traits, respectively. These nutrigenetic findings indicate genotype can modify the associations between vegetarianism and health outcomes.

Correction: Genome-wide association study of fish oil supplementation on lipid traits in 81,246 individuals reveals new gene-diet interaction loci.

[This corrects the article DOI: 10.1371/journal.pgen.1009431.].

Whole-exome sequencing study identifies four novel gene loci associated with diabetic kidney disease.

Diabetic kidney disease (DKD) is recognized as an important public health challenge. However, its genomic mechanisms are poorly understood. To identify rare variants for DKD, we conducted a whole-exome sequencing (WES) study leveraging large cohorts well-phenotyped for chronic kidney disease and diabetes. Our two-stage WES study included 4372 European and African ancestry participants from the Chronic Renal Insufficiency Cohort and Atherosclerosis Risk in Communities studies (stage 1) and 11 487 multi-ancestry Trans-Omics for Precision Medicine participants (stage 2). Generalized linear mixed models, which accounted for genetic relatedness and adjusted for age, sex and ancestry, were used to test associations between single variants and DKD. Gene-based aggregate rare variant analyses were conducted using an optimized sequence kernel association test implemented within our mixed model framework. We identified four novel exome-wide significant DKD-related loci through initiating diabetes. In single-variant analyses, participants carrying a rare, in-frame insertion in the DIS3L2 gene (rs141560952) exhibited a 193-fold increased odds [95% confidence interval (CI): 33.6, 1105] of DKD compared with noncarriers (P = 3.59 × 10-9). Likewise, each copy of a low-frequency KRT6B splice-site variant (rs425827) conferred a 5.31-fold higher odds (95% CI: 3.06, 9.21) of DKD (P = 2.72 × 10-9). Aggregate gene-based analyses further identified ERAP2 (P = 4.03 × 10-8) and NPEPPS (P = 1.51 × 10-7), which are both expressed in the kidney and implicated in renin-angiotensin-aldosterone system modulated immune response. In the largest WES study of DKD, we identified novel rare variant loci attaining exome-wide significance. These findings provide new insights into the molecular mechanisms underlying DKD.

Hunting Drosophila viruses from wild populations: A novel isolation approach and characterisation of viruses.

Metagenomic studies have demonstrated that viruses are extremely diverse and abundant in insects, but the difficulty of isolating them means little is known about the biology of these newly discovered viruses. To overcome this challenge in Drosophila, we created a cell line that was more permissive to infection and detected novel viruses by the presence of double-stranded RNA. We demonstrate the utility of these tools by isolating La Jolla virus (LJV) and Newfield virus (NFV) from several wild Drosophila populations. These viruses have different potential host ranges, with distinct abilities to replicate in five Drosophila species. Similarly, in some species they cause high mortality and in others they are comparatively benign. In three species, NFV but not LJV caused large declines in female fecundity. This sterilization effect was associated with differences in tissue tropism, as NFV but not LJV was able to infect Drosophila melanogaster follicular epithelium and induce follicular degeneration in the ovary. We saw a similar effect in the invasive pest of fruit crops Drosophila suzukii, where oral infection with NFV caused reductions in the fecundity, suggesting it has potential as a biocontrol agent. In conclusion, a simple protocol allowed us to isolate new viruses and demonstrate that viruses identified by metagenomics have a large effect on the fitness of the model organism D. melanogaster and related species.

Correction: Kinesin-3 mediated axonal delivery of presynaptic neurexin stabilizes dendritic spines and postsynaptic components.

[This corrects the article DOI: 10.1371/journal.pgen.1010016.].

Kinesin-3 mediated axonal delivery of presynaptic neurexin stabilizes dendritic spines and postsynaptic components.

The functional properties of neural circuits are defined by the patterns of synaptic connections between their partnering neurons, but the mechanisms that stabilize circuit connectivity are poorly understood. We systemically examined this question at synapses onto newly characterized dendritic spines of C. elegans GABAergic motor neurons. We show that the presynaptic adhesion protein neurexin/NRX-1 is required for stabilization of postsynaptic structure. We find that early postsynaptic developmental events proceed without a strict requirement for synaptic activity and are not disrupted by deletion of neurexin/nrx-1. However, in the absence of presynaptic NRX-1, dendritic spines and receptor clusters become destabilized and collapse prior to adulthood. We demonstrate that NRX-1 delivery to presynaptic terminals is dependent on kinesin-3/UNC-104 and show that ongoing UNC-104 function is required for postsynaptic maintenance in mature animals. By defining the dynamics and temporal order of synapse formation and maintenance events in vivo, we describe a mechanism for stabilizing mature circuit connectivity through neurexin-based adhesion.

Genome-wide association study of fish oil supplementation on lipid traits in 81,246 individuals reveals new gene-diet interaction loci.

Fish oil supplementation is widely used for reducing serum triglycerides (TAGs) but has mixed effects on other circulating cardiovascular biomarkers. Many genetic polymorphisms have been associated with blood lipids, including high- and low-density-lipoprotein cholesterol (HDL-C, LDL-C), total cholesterol, and TAGs. Here, the gene-diet interaction effects of fish oil supplementation on these lipids were analyzed in a discovery cohort of up to 73,962 UK Biobank participants, using a 1-degree-of-freedom (1df) test for interaction effects and a 2-degrees-of-freedom (2df) test to jointly analyze interaction and main effects. Associations with P < 1×10-6 in either test (26,157; 18,300 unique variants) were advanced to replication in up to 7,284 participants from the Atherosclerosis Risk in Communities (ARIC) Study. Replicated associations reaching 1df P < 0.05 (2,175; 1,763 unique variants) were used in meta-analyses. We found 13 replicated and 159 non-replicated (UK Biobank only) loci with significant 2df joint tests that were predominantly driven by main effects and have been previously reported. Four novel interaction loci were identified with 1df P < 5×10-8 in meta-analysis. The lead variant in the GJB6-GJB2-GJA3 gene cluster, rs112803755 (A>G; minor allele frequency = 0.041), shows exclusively interaction effects. The minor allele is significantly associated with decreased TAGs in individuals with fish oil supplementation, but with increased TAGs in those without supplementation. This locus is significantly associated with higher GJB2 expression of connexin 26 in adipose tissue; connexin activity is known to change upon exposure to omega-3 fatty acids. Significant interaction effects were also found in three other loci in the genes SLC12A3 (HDL-C), ABCA6 (LDL-C), and MLXIPL (LDL-C), but highly significant main effects are also present. Our study identifies novel gene-diet interaction effects for four genetic loci, whose effects on blood lipids are modified by fish oil supplementation. These findings highlight the need and possibility for personalized nutrition.

Editorial Commentary: Biocomposite Implant Combined With Local Human Recombinant Parathyroid Hormone Enhances the Histological Quality and Biomechanical Strength of Rotator Cuff Repairs.

Rates of rotator cuff repair retear remain unacceptably high and are frequently the source of diminished shoulder function and patient dissatisfaction. Endocrinopathies have been implicated in these processes. Parathyroid hormone (PTH) activates chondrogenesis and angiogenesis at the enthesis and prevents fatty infiltration and atrophy in rotator cuff musculature. These facts have spurred interest in the therapeutic benefits of PTH as a means to enhance tendon healing and strengthen the bone in and around tendon repairs. New research demonstrates that recombinant human PTH delivered locally through a process of coupling it to a bioengineered scaffold "sheath" may be beneficial. The growth factor, encased within polycaprolactone (PCL), is slowly released as the PCL degrades to extend drug delivery time. The augmentation of rotator cuff repairs with this biocomposite material improves short-term structural tissue integrity and promotes the formation of more organized and stronger tendon-to-bone interface in a rabbit model.

Considerations for rapid development and licencing of conventional and platform technology veterinary vaccines.

The threat posed by zoonotic diseases and other livestock pathogens has never been greater, and thus we must do all we can to learn from experience in order to tackle emerging disease threats. The process of developing a new veterinary vaccine involves the generation of a specific set of data in order to meet the strict product licencing requirements of regulatory approval bodies around the globe. As a result, it is important that those embarking on the development of a vaccine using either conventional or novel platform technologies understand these regulations. In addition, there are a number of specific requirements that one needs to take into consideration when developing a product specifically for the commercial poultry market. This paper briefly outlines the veterinary vaccine development process in general and then explores how this process can be accelerated. It also recognizes the "One Health" lessons that can be learnt from the recent rapid development of vaccines to tackle the COVID-19 pandemic and acknowledges the important measures that regulatory authorities have taken in the creation of an environment to facilitate the licencing of new vaccine platform technologies.

The effects of recombinant human insulin-like growth factor-1/insulin-like growth factor binding protein-3 administration on lipid and carbohydrate metabolism in recreational athletes.

OBJECTIVE: Previous studies suggested that recombinant human IGF-1 (rhIGF-1) administration affects carbohydrate and lipid metabolism in healthy people and in people with diabetes. This study aimed to determine the effects of rhIGF-1/rhIGF binding protein-3 (rhIGFBP-3) administration on glucose homeostasis and lipid metabolism in healthy recreational athletes. DESIGN AND SETTING: Randomized, double-blind, placebo-controlled rhIGF-1/rhIGFBP-3 administration study at Southampton General Hospital, UK. PARTICIPANTS: 56 recreational athletes (30 men, 26 women). METHODS: Participants were randomly assigned to receive placebo, low-dose rhIGF-1/rhIGFBP-3 (30 mg/day) or high-dose rhIGF-1/rhIGFBP-3 (60 mg/day) for 28 days. The following variables were measured before and immediately after the treatment period: fasting lipids, glucose, insulin, C-peptide and glycated haemoglobin. The homeostatic model assessment (HOMA-IR) was used to estimate insulin sensitivity and indirect calorimetry to assess substrate oxidation rates. The general linear model approach was used to compare treatment group changes with the placebo group. RESULTS: Compared with the placebo group, there was a significant reduction in fasting triglycerides in participants treated with high-dose rhIGF-1/rhIGFBP-3 (p = .030), but not in the low-dose group (p = .390). In women, but not in men, there were significant increases in total cholesterol (p = .003), HDL cholesterol (p = .001) and LDL cholesterol (p = .008). These lipid changes were associated with reduced fasting insulin (p = .010), C-peptide (p = .001) and HOMA-IR (p = .018) in women and reduced C-peptide (p = .046) in men. CONCLUSIONS: rhIGF-1/rhIGFBP-3 administration for 28 days reduced insulin concentration, improved insulin sensitivity and had significant effects on lipid profile including decreased fasting triglycerides.

Spotlight on avian pathology: the importance of recombinant vector platform technologies in poultry vaccination.

The use of novel vector vaccines (viral, bacterial and apicomplexan) can have a significant impact on the control of poultry disease. They offer a cost effective, convenient and effective means of mass vaccine delivery combined with the ability to switch on both antibody and cell-mediated immunity. In addition, recent viral vector constructs have enabled farmers to vaccinate against up to three important pathogens with a single in ovo administration. As the technology develops, it is likely that this means of vaccine administration will be utilized further and it will play a key role in the control of both existing and new emerging diseases of poultry in the future.

Role of the global regulator Rex in control of NAD+ -regeneration in Clostridioides (Clostridium) difficile.

For the human pathogen Clostridioides (also known as Clostridium) difficile, the ability to adapt to nutrient availability is critical for its proliferation and production of toxins during infection. Synthesis of the toxins is regulated by the availability of certain carbon sources, fermentation products and amino acids (e.g. proline, cysteine, isoleucine, leucine and valine). The effect of proline is attributable at least in part to its role as an inducer and substrate of D-proline reductase (PR), a Stickland reaction that regenerates NAD+ from NADH. Many Clostridium spp. use Stickland metabolism (co-fermentation of pairs of amino acids) to generate ATP and NAD+ . Synthesis of PR is activated by PrdR, a proline-responsive regulatory protein. Here we report that PrdR, in the presence of proline, represses other NAD+ -generating pathways, such as the glycine reductase and succinate-acetyl CoA utilization pathways leading to butyrate production, but does so indirectly by affecting the activity of Rex, a global redox-sensing regulator that responds to the NAD+ /NADH ratio. Our results indicate that PR activity is the favored mechanism for NAD+ regeneration and that both Rex and PrdR influence toxin production. Using the hamster model of C. difficile infection, we revealed the importance of PrdR-regulated Stickland metabolism in the virulence of C. difficile.

Excitatory neurons sculpt GABAergic neuronal connectivity in the C. elegans motor circuit.

Establishing and maintaining the appropriate number of GABA synapses is key for balancing excitation and inhibition in the nervous system, though we have only a limited understanding of the mechanisms controlling GABA circuit connectivity. Here, we show that disrupting cholinergic innervation of GABAergic neurons in the C. elegans motor circuit alters GABAergic neuron synaptic connectivity. These changes are accompanied by reduced frequency and increased amplitude of GABAergic synaptic events. Acute genetic disruption in early development, during the integration of post-embryonic-born GABAergic neurons into the circuit, produces irreversible effects on GABAergic synaptic connectivity that mimic those produced by chronic manipulations. In contrast, acute genetic disruption of cholinergic signaling in the adult circuit does not reproduce these effects. Our findings reveal that GABAergic signaling is regulated by cholinergic neuronal activity, probably through distinct mechanisms in the developing and mature nervous system.

Local neuropeptide signaling modulates serotonergic transmission to shape the temporal organization of C. elegans egg-laying behavior.

Animal behaviors are often composed of distinct alternating behavioral states. Neuromodulatory signals are thought to be critical for establishing stable behavioral states and for orchestrating transitions between them. However, we have only a limited understanding of how neuromodulatory systems act in vivo to alter circuit performance and shape behavior. To address these questions, we have investigated neuromodulatory signaling in the context of Caenorhabditis elegans egg-laying. Egg-laying activity cycles between discrete states-short bursts of egg deposition (active phases) that alternate with prolonged quiescent periods (inactive phases). Here using genetic, pharmacological and optogenetic approaches for cell-specific activation and inhibition, we show that a group of neurosecretory cells (uv1) located in close spatial proximity to the egg-laying neuromusculature direct the temporal organization of egg-laying by prolonging the duration of inactive phases. We demonstrate that the modulatory effects of the uv1 cells are mediated by peptides encoded by the nlp-7 and flp-11 genes that act locally to inhibit circuit activity, primarily by inhibiting vesicular release of serotonin from HSN motor neurons. This peptidergic inhibition is achieved, at least in part, by reducing synaptic vesicle abundance in the HSN motor neurons. By linking the in vivo actions of specific neuropeptide signaling systems with the generation of stable behavioral outcomes, our study reveals how cycles of neuromodulation emanating from non-neuronal cells can fundamentally shape the organization of a behavioral program.

MiRNA-target interactions in osteogenic signaling pathways involving zinc via the metal regulatory element.

Adequate zinc nutriture is necessary for normal bone growth and development, though the precise mechanisms for zinc-mediated bone growth remain poorly defined. A key transcription factor activated by zinc is metal response element-binding transcription factor 1 (MTF-1), which binds to the metal regulatory element (MRE). We hypothesize that MREs will be found upstream of miRNA genes as well as miRNA target genes in the following bone growth and development signaling pathways: TGF-ß, MAPK, and Wnt. A Bioconductor-based workflow in R was designed to identify interactions between MREs, miRNAs, and target genes. MRE sequences were found upstream from 64 mature miRNAs that interact with 213 genes which have MRE sequences in their own promoter regions. MAPK1 exhibited the most miRNA-target interactions (MTIs) in the TGF-ß and MAPK signaling pathways; CCND2 exhibited the most interactions in the Wnt signaling pathway. Hsa-miR-124-3p exhibited the most MTIs in the TGF-ß and MAPK signaling pathways; hsa-miR-20b-5p exhibited the most MTIs in the Wnt signaling pathway. MYC and hsa-miR-34a-5p were shared between all three signaling pathways, also forming an MTI unit. JUN exhibited the most protein-protein interactions, followed by MAPK8. These in silico data support the hypothesis that intracellular zinc status plays a role in osteogenesis through the transcriptional regulation of miRNA genes via the zinc/MTF-1/MRE complex.

The Effects of Bone Microstructure on Subsidence Risk for ALIF, LLIF, PLIF, and TLIF Spine Cages.

Several approaches (anterior, posterior, lateral, and transforaminal) are used in lumbar fusion surgery. However, it is unclear whether one of these approaches has the greatest subsidence risk as published clinical rates of cage subsidence vary widely (7-70%). Specifically, there is limited data on how a patient's endplate morphometry and trabecular bone quality influences cage subsidence risk. Therefore, this study compared subsidence (stiffness, maximum force, and work) between anterior (ALIF), lateral (LLIF), posterior (PLIF), and transforaminal (TLIF) lumbar interbody fusion cage designs to understand the impact of endplate and trabecular bone quality on subsidence. Forty-eight lumbar vertebrae were imaged with micro-ct to assess trabecular microarchitecture. micro-ct images of each vertebra were then imported into image processing software to measure endplate thickness (ET) and maximum endplate concavity depth (ECD). Generic ALIF, LLIF, PLIF, and TLIF cages made of polyether ether ketone were implanted on the superior endplates of all vertebrae and subsidence testing was performed. The results indicated that TLIF cages had significantly lower (p < 0.01) subsidence stiffness and maximum subsidence force compared to ALIF and LLIF cages. For all cage groups, trabecular bone volume fraction was better correlated with maximum subsidence force compared to ET and concavity depth. These findings highlight the importance of cage design (e.g., surface area), placement on the endplate, and trabecular bone quality on subsidence. These results may help surgeons during cage selection for lumbar fusion procedures to mitigate adverse events such as cage subsidence.

Use of clinical quality indicators to improve lung cancer care in a regional/rural network of health services.

PROBLEM: Optimal lung cancer care requires multidisciplinary team input, with access to specialised diagnostic and therapeutic services that may be limited in rural or regional areas and impact clinical outcomes. Clinical quality indicators can be used to measure the quality of care delivered to patients with lung cancer in a region and identify areas for improvement. We describe the implementation of internationally recognised clinical quality indicators for lung cancer care in the Barwon South Western region. DESIGN: The consensus of an expert panel was used for the selection of clinical quality indicators. The data were retrospectively collected from the Evaluation of Cancer Outcomes Barwon South West Registry, which systematically records detailed information on all new patients with cancer in the region. SETTING: Region-based health service. KEY MEASURES FOR IMPROVEMENT: Adherence to clinical quality indicator targets. STRATEGIES FOR CHANGE: Clinical quality indicators, which fall short of the expected targets, highlight areas for improvement in the service provided to patients with lung cancer. These results have prompted changes in the service offered to these patients, such as the introduction of a multidisciplinary lung cancer clinic. EFFECTS OF CHANGE: The multidisciplinary lung cancer clinic has streamlined the access to lung cancer services, including specialist consultations, diagnostics and therapeutic services, in a regional setting. Ongoing data collection is required to determine the effect of such changes on adherence to clinical quality indicator targets. LESSONS LEARNT: The regular monitoring of clinical quality indicators serves as a useful method of quality assurance in the care of patients with lung cancer. We expect these clinical quality indicators to also be used by other health services to analyse and improve services provided to patients with lung cancer.

Long-term high-altitude hypoxia influences pulmonary arterial L-type calcium channel-mediated Ca2+ signals and contraction in fetal and adult sheep.

Long-term hypoxia (LTH) has a profound effect on pulmonary arterial vasoconstriction in the fetus and adult. Dysregulation in Ca2+ signaling is important during the development of LTH-induced pulmonary hypertension. In the present study, we tested the hypothesis that L-type Ca2+ channels (CaL), which are voltage dependent and found in smooth, skeletal, and cardiac muscle, are important in the adaptation of pulmonary arterial contractions in postnatal maturation and in response to LTH. Pulmonary arteries were isolated from fetal or adult sheep maintained at low or high altitude (3,801 m) for >100 days. The effects were measured using an L-type Ca2+ channel opener FPL 64176 (FPL) in the presence or absence of an inhibitor, Nifedipine (NIF) on arterial contractions, intracellular Ca2+ oscillations, and ryanodine receptor-driven Ca2+ sparks. FPL induced pulmonary arterial contractions in all groups were sensitive to NIF. However, when compared with 125 mM K+, FPL contractions were greater in fetuses than in adults. FPL reduced Ca2+ oscillations in myocytes of adult but not fetal arteries, independently of altitude. The FPL effects on Ca2+ oscillations were reversed by NIF in myocytes of hypoxic but not normoxic adults. FPL failed to enhance Ca2+ spark frequency and had little impact on spatiotemporal firing characteristics. These data suggest that CaL-dependent contractions are largely uncoupled from intracellular Ca2+ oscillations and the development of Ca2+ sparks. This raises questions regarding the coupling of pulmonary arterial contractility to membrane depolarization, attendant CaL facilitation, and the related associations with the activation of Ca2+ oscillations and Ca2+ sparks.

Functional Tuning of Intrinsic Endothelial Ca2+ Dynamics in Swine Coronary Arteries.

RATIONALE: Recent data from mesenteric and cerebral beds have revealed spatially restricted Ca(2+) transients occurring along the vascular intima that control effector recruitment and vasodilation. Although Ca(2+) is pivotal for coronary artery endothelial function, spatial and temporal regulation of functional Ca(2+) signals in the coronary endothelium is poorly understood. OBJECTIVE: We aimed to determine whether a discrete spatial and temporal profile of Ca(2+) dynamics underlies endothelium-dependent relaxation of swine coronary arteries. METHODS AND RESULTS: Using confocal imaging, custom automated image analysis, and myography, we show that the swine coronary artery endothelium generates discrete basal Ca(2+) dynamics, including isolated transients and whole-cell propagating waves. These events are suppressed by depletion of internal stores or inhibition of inositol 1,4,5-trisphosphate receptors but not by inhibition of ryanodine receptors or removal of extracellular Ca(2+). In vessel rings, inhibition of specific Ca(2+)-dependent endothelial effectors, namely, small and intermediate conductance K(+) channels (K(Ca)3.1 and K(Ca)2.3) and endothelial nitric oxide synthase, produces additive tone, which is blunted by internal store depletion or inositol 1,4,5-trisphosphate receptor blockade. Stimulation of endothelial inositol 1,4,5-trisphosphate-dependent signaling with substance P causes idiosyncratic changes in dynamic Ca(2+) signal parameters (active sites, event frequency, amplitude, duration, and spatial spread). Overall, substance P-induced vasorelaxation corresponded poorly with whole-field endothelial Ca(2+) measurements but corresponded precisely with the concentration-dependent change in Ca(2+) dynamics (linearly translated composite of dynamic parameters). CONCLUSIONS: Our findings show that endothelium-dependent control of swine coronary artery tone is determined by spatial and temporal titration of inherent endothelial Ca(2+) dynamics that are not represented by tissue-level averaged Ca(2+) changes.