Angiotensin II Directly Increases Endothelial Calcium and Nitric Oxide in Kidney and Brain Microvessels In Vivo With Reduced Efficacy in Hypertension.

BACKGROUND: The vasoconstrictor effects of angiotensin II via type 1 angiotensin II receptors in vascular smooth muscle cells are well established, but the direct effects of angiotensin II on vascular endothelial cells (VECs) in vivo and the mechanisms how VECs may mitigate angiotensin II-mediated vasoconstriction are not fully understood. The present study aimed to explore the molecular mechanisms and pathophysiological relevance of the direct actions of angiotensin II on VECs in kidney and brain microvessels in vivo. METHODS AND RESULTS: Changes in VEC intracellular calcium ([Ca2+]i) and nitric oxide (NO) production were visualized by intravital multiphoton microscopy of cadherin 5-Salsa6f mice or the endothelial uptake of NO-sensitive dye 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, respectively. Kidney fibrosis by unilateral ureteral obstruction and Ready-to-use adeno-associated virus expressing Mouse Renin 1 gene (Ren1-AAV) hypertension were used as disease models. Acute systemic angiotensin II injections triggered >4-fold increases in VEC [Ca2+]i in brain and kidney resistance arterioles and capillaries that were blocked by pretreatment with the type 1 angiotensin II receptor inhibitor losartan, but not by the type 2 angiotensin II receptor inhibitor PD123319. VEC responded to acute angiotensin II by increased NO production as indicated by >1.5-fold increase in 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate fluorescence intensity. In mice with kidney fibrosis or hypertension, the angiotensin II-induced VEC [Ca2+]i and NO responses were significantly reduced, which was associated with more robust vasoconstrictions, VEC shedding, and microthrombi formation. CONCLUSIONS: The present study directly visualized angiotensin II-induced increases in VEC [Ca2+]i and NO production that serve to counterbalance agonist-induced vasoconstriction and maintain residual organ blood flow. These direct and endothelium-specific angiotensin II effects were blunted in disease conditions and linked to endothelial dysfunction and the development of vascular pathologies.

Enriched Single-Nucleus RNA-Sequencing Reveals Unique Attributes of Distal Convoluted Tubule Cells.

SIGNIFICANCE STATEMENT: High-resolution single-nucleus RNA-sequencing data indicate a clear separation between primary sites of calcium and magnesium handling within distal convoluted tubule (DCT). Both DCT1 and DCT2 express Slc12a3, but these subsegments serve distinctive functions, with more abundant magnesium-handling genes along DCT1 and more calcium-handling genes along DCT2. The data also provide insight into the plasticity of the distal nephron-collecting duct junction, formed from cells of separate embryonic origins. By focusing/changing gradients of gene expression, the DCT can morph into different physiological cell states on demand. BACKGROUND: The distal convoluted tubule (DCT) comprises two subsegments, DCT1 and DCT2, with different functional and molecular characteristics. The functional and molecular distinction between these segments, however, has been controversial. METHODS: To understand the heterogeneity within the DCT population with better clarity, we enriched for DCT nuclei by using a mouse line combining "Isolation of Nuclei Tagged in specific Cell Types" and sodium chloride cotransporter-driven inducible Cre recombinase. We sorted the fluorescently labeled DCT nuclei using Fluorescence-Activated Nucleus Sorting and performed single-nucleus transcriptomics. RESULTS: Among 25,183 DCT cells, 75% were from DCT1 and 25% were from DCT2. In addition, there was a small population (<1%) enriched in proliferation-related genes, such as Top2a , Cenpp , and Mki67 . Although both DCT1 and DCT2 expressed sodium chloride cotransporter, magnesium transport genes were predominantly expressed along DCT1, whereas calcium, electrogenic sodium, and potassium transport genes were more abundant along DCT2. The transition between these two segments was gradual, with a transitional zone in which DCT1 and DCT2 cells were interspersed. The expression of the homeobox genes by DCT cells suggests that they develop along different trajectories. CONCLUSIONS: Transcriptomic analysis of an enriched rare cell population using a genetically targeted approach clarifies the function and classification of distal cells. The DCT segment is short, can be separated into two subsegments that serve distinct functions, and is speculated to derive from different origins during development.

COP9 signalosome deletion promotes renal injury and distal convoluted tubule remodeling.

Cullin-RING ligases are a family of E3 ubiquitin ligases that control cellular processes through regulated degradation. Cullin 3 targets with-no-lysine kinase 4 (WNK4), a kinase that activates the Na+-Cl- cotransporter (NCC), the main pathway for Na+ reabsorption in the distal convoluted tubule (DCT). Mutations in the cullin 3 gene lead to familial hyperkalemic hypertension by increasing WNK4 abundance. The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) regulates the activity of cullin-RING ligases by removing the ubiquitin-like protein neural precursor cell expressed developmentally downregulated protein 8. Genetic deletion of the catalytically active CSN subunit, Jab1, along the nephron in mice (KS-Jab1-/-) led to increased WNK4 abundance; however, NCC abundance was substantially reduced. We hypothesized that the reduction in NCC resulted from a cortical injury that led to hypoplasia of the segment, which counteracted WNK4 activation of NCC. To test this, we studied KS-Jab1-/- mice at weekly intervals over a period of 3 wk. The results showed that NCC abundance was unchanged until 3 wk after Jab1 deletion, at which time other DCT-specific proteins were also reduced. The kidney injury markers kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin demonstrated kidney injury immediately after Jab1 deletion; however, the damage was initially limited to the medulla. The injury progressed and expanded into the cortex 3 wk after Jab1 deletion coinciding with loss of the DCT. The data indicate that nephron-specific disruption of the cullin-RING ligase system results in a complex progression of tubule injury that leads to hypoplasia of the DCT.NEW & NOTEWORTHY Cullin 3 (CUL3) targets with-no-lysine-kinase 4 (WNK4), which activates Na+-Cl- cotransporter (NCC) in the distal convoluted tubule (DCT) of the kidney. Renal-specific genetic deletion of the constitutive photomorphogenesis 9 signalosome, an upstream regulator of CUL3, resulted in a reduction of NCC due to DCT hypoplasia, which coincided with cortical kidney injury. The data indicate that nephron-specific disruption of the cullin-RING ligase system results in a complex progression of tubule injury leading to hypoplasia of the DCT.

Control of coronary vascular resistance by eicosanoids via a novel GPCR.

Arachidonic acid metabolites epoxyeicosatrienoates (EETs) and hydroxyeicosatetraenoates (HETEs) are important regulators of myocardial blood flow and coronary vascular resistance (CVR), but their mechanisms of action are not fully understood. We applied a chemoproteomics strategy using a clickable photoaffinity probe to identify G protein-coupled receptor 39 (GPR39) as a microvascular smooth muscle cell (mVSMC) receptor selective for two endogenous eicosanoids, 15-HETE and 14,15-EET, which act on the receptor to oppose each other's activity. The former increases mVSMC intracellular calcium via GPR39 and augments coronary microvascular resistance, and the latter inhibits these actions. Furthermore, we find that the efficacy of both ligands is potentiated by zinc acting as an allosteric modulator. Measurements of coronary perfusion pressure (CPP) in GPR39-null hearts using the Langendorff preparation indicate the receptor senses these eicosanoids to regulate microvascular tone. These results implicate GPR39 as an eicosanoid receptor and key regulator of myocardial tissue perfusion. Our findings will have a major impact on understanding the roles of eicosanoids in cardiovascular physiology and disease and provide an opportunity for the development of novel GPR39-targeting therapies for cardiovascular disease.

Soluble ACE2 Is Filtered into the Urine.

Background: ACE2 is a key enzyme in the renin-angiotensin system (RAS) capable of balancing the RAS by metabolizing angiotensin II (AngII). First described in cardiac tissue, abundance of ACE2 is highest in the kidney, and it is also expressed in several extrarenal tissues. Previously, we reported an association between enhanced susceptibility to hypertension and elevated renal AngII levels in global ACE2-knockout mice. Methods: To examine the effect of ACE2 expressed in the kidney, relative to extrarenal expression, on the development of hypertension, we used a kidney crosstransplantation strategy with ACE2-KO and WT mice. In this model, both native kidneys are removed and renal function is provided entirely by the transplanted kidney, such that four experimental groups with restricted ACE2 expression are generated: WT→WT (WT), KO→WT (KidneyKO), WT→KO (SystemicKO), and KO→KO (TotalKO). Additionally, we used nanoscale mass spectrometry-based proteomics to identify ACE2 fragments in early glomerular filtrate of mice. Results: Although significant differences in BP were not detected, a major finding of our study is that shed or soluble ACE2 (sACE2) was present in urine of KidneyKO mice that lack renal ACE2 expression. Detection of sACE2 in the urine of KidneyKO mice during AngII-mediated hypertension suggests that sACE2 originating from extrarenal tissues can reach the kidney and be excreted in urine. To confirm glomerular filtration of ACE2, we used micropuncture and nanoscale proteomics to detect peptides derived from ACE2 in the Bowman's space. Conclusions: Our findings suggest that both systemic and renal tissues may contribute to sACE2 in urine, identifying the kidney as a major site for ACE2 actions. Moreover, filtration of sACE2 into the lumen of the nephron may contribute to the pathophysiology of kidney diseases characterized by disruption of the glomerular filtration barrier.

GPR39 localization in the aging human brain and correlation of expression and polymorphism with vascular cognitive impairment.

INTRODUCTION: The pathogenesis of vascular cognitive impairment (VCI) is not fully understood. GPR39, an orphan G-protein coupled receptor, is implicated in neurological disorders but its role in VCI is unknown. METHODS: We performed GPR39 immunohistochemical analysis in post mortem brain samples from mild cognitive impairment (MCI) and control subjects. DNA was analyzed for GPR39 single nucleotide polymorphisms (SNPs), and correlated with white matter hyperintensity (WMH) burden on pre mortem magnetic resonance imaging. RESULTS: GPR39 is expressed in aged human dorsolateral prefrontal cortex, localized to microglia and peri-capillary cells resembling pericytes. GPR39-capillary colocalization, and density of GPR39-expressing microglia was increased in aged brains compared to young. SNP distribution was equivalent between groups; however, homozygous SNP carriers were present only in the MCI group, and had higher WMH volume than wild-type or heterozygous SNP carriers. DISCUSSION: GPR39 may play a role in aging-related VCI, and may serve as a therapeutic target and biomarker for the risk of developing VCI.

Age-dependent transcriptional alterations in cardiac endothelial cells.

Aging is a significant risk factor for cardiovascular disease. Despite the fact that endothelial cells play critical roles in cardiovascular function and disease, the molecular impact of aging on this cell population in many organ systems remains unknown. In this study, we sought to determine age-associated transcriptional alterations in cardiac endothelial cells. Highly enriched populations of endothelial cells (ECs) isolated from the heart, brain, and kidney of young (3 mo) and aged (24 mo) C57/BL6 mice were profiled for RNA expression via bulk RNA sequencing. Approximately 700 cardiac endothelial transcripts significantly differ by age. Gene set enrichment analysis indicated similar patterns for cellular pathway perturbations. Receptor-ligand comparisons indicated parallel alterations in age-affected circulating factors and cardiac endothelial-expressed receptors. Gene and pathway enrichment analyses show that age-related transcriptional response of cardiac endothelial cells is distinct from that of endothelial cells derived from the brain or kidney vascular bed. Furthermore, single-cell analysis identified nine distinct EC subtypes and shows that the Apelin Receptor-enriched subtype is reduced with age in mouse heart. Finally, we identify age-dysregulated genes in specific aged cardiac endothelial subtypes.

Local and downstream actions of proximal tubule angiotensin II signaling on Na+ transporters in the mouse nephron.

The renal nephron consists of a series of distinct cell types that function in concert to maintain fluid and electrolyte balance and blood pressure. The renin-angiotensin system (RAS) is central to Na+ and volume balance. We aimed to determine how loss of angiotensin II signaling in the proximal tubule (PT), which reabsorbs the bulk of filtered Na+ and volume, impacts solute transport throughout the nephron. We hypothesized that PT renin-angiotensin system disruption would not only depress PT Na+ transporters but also impact downstream Na+ transporters. Using a mouse model in which the angiotensin type 1a receptor (AT1aR) is deleted specifically within the PT (AT1aR PTKO), we profiled the abundance of Na+ transporters, channels, and claudins along the nephron. Absence of PT AT1aR signaling was associated with lower abundance of PT transporters (Na+/H+ exchanger isoform 3, electrogenic Na+-bicarbonate cotransporter 1, and claudin 2) as well as lower abundance of downstream transporters (total and phosphorylated Na+-K+-2Cl- cotransporter, medullary Na+-K+-ATPase, phosphorylated NaCl cotransporter, and claudin 7) versus controls. However, transport activities of Na+-K+-2Cl- cotransporter and NaCl cotransporter (assessed with diuretics) were similar between groups in order to maintain electrolyte balance. Together, these results demonstrate the primary impact of angiotensin II regulation on Na+ reabsorption in the PT at baseline and the associated influence on downstream Na+ transporters, highlighting the ability of the nephron to integrate Na+ transport along the nephron to maintain homeostasis.NEW & NOTEWORTHY Our study defines a novel role for proximal tubule angiotensin receptors in regulating the abundance of Na+ transporters throughout the nephron, thereby contributing to the integrated control of fluid balance in vivo.

Advances in use of mouse models to study the renin-angiotensin system.

The renin-angiotensin system (RAS) is a highly complex hormonal cascade that spans multiple organs and cell types to regulate solute and fluid balance along with cardiovascular function. Much of our current understanding of the functions of the RAS has emerged from a series of key studies in genetically-modified animals. Here, we review key findings from ground-breaking transgenic models, spanning decades of research into the RAS, with a focus on their use in studying blood pressure. We review the physiological importance of this regulatory system as evident through the examination of mouse models for several major RAS components: angiotensinogen, renin, ACE, ACE2, and the type 1 A angiotensin receptor. Both whole-animal and cell-specific knockout models have permitted critical RAS functions to be defined and demonstrate how redundancy and multiplicity within the RAS allow for compensatory adjustments to maintain homeostasis. Moreover, these models present exciting opportunities for continued discovery surrounding the role of the RAS in disease pathogenesis and treatment for cardiovascular disease and beyond.

Mg2+ restriction downregulates NCC through NEDD4-2 and prevents its activation by hypokalemia.

Hypomagnesemia is associated with reduced kidney function and life-threatening complications and sustains hypokalemia. The distal convoluted tubule (DCT) determines final urinary Mg2+ excretion and, via activity of the Na+-Cl- cotransporter (NCC), also plays a key role in K+ homeostasis by metering Na+ delivery to distal segments. Little is known about the mechanisms by which plasma Mg2+ concentration regulates NCC activity and how low-plasma Mg2+ concentration and K+ concentration interact to modulate NCC activity. To address this, we performed dietary manipulation studies in mice. Compared with normal diet, abundances of total NCC and phosphorylated NCC (pNCC) were lower after short-term (3 days) or long-term (14 days) dietary Mg2+ restriction. Altered NCC activation is unlikely to play a role, since we also observed lower total NCC abundance in mice lacking the two NCC-activating kinases, STE20/SPS-1-related proline/alanine-rich kinase and oxidative stress response kinase-1, after Mg2+ restriction. The E3 ubiquitin-protein ligase NEDD4-2 regulates NCC abundance during dietary NaCl loading or K+ restriction. Mg2+ restriction did not lower total NCC abundance in inducible nephron-specific neuronal precursor cell developmentally downregulated 4-2 (NEDD4-2) knockout mice. Total NCC and pNCC abundances were similar after short-term Mg2+ or combined Mg2+-K+ restriction but were dramatically lower compared with a low-K+ diet. Therefore, sustained NCC downregulation may serve a mechanism that enhances distal Na+ delivery during states of hypomagnesemia, maintaining hypokalemia. Similar results were obtained with long-term Mg2+-K+ restriction, but, surprisingly, NCC was not activated after long-term K+ restriction despite lower plasma K+ concentration, suggesting significant differences in distal tubule adaptation to acute or chronic K+ restriction.

Cullin-Ring ubiquitin ligases in kidney health and disease.

PURPOSE OF REVIEW: Members of the Cullin family act as scaffolds in E3 ubiquitin ligases and play a central role in mediating protein degradation. Interactions with many different substrate-binding adaptors permit Cullin-containing E3 ligases to participate in diverse cellular functions. In the kidney, one well established target of Cullin-mediated degradation is the transcription factor Nrf2, a key player in responses to oxidative stress. The goal of this review is to discuss more recent findings revealing broader roles for Cullins in the kidney. RECENT FINDINGS: Cullin 3 acts as the scaffold in the E3 ligase regulating Nrf2 abundance, but was more recently shown to be mutated in the disease familial hyperkalemic hypertension. Studies seeking to elucidate the molecular mechanisms by which Cullin 3 mutations lead to dysregulation of renal sodium transport will be discussed. Disruption of Cullin 3 in mice unexpectedly causes polyuria and fibrotic injury suggesting it has additional roles in the kidney. We will also review recent transcriptomic data suggesting that other Cullins are also likely to play important roles in renal function. SUMMARY: Cullins form a large and diverse family of E3 ubiquitin ligases that are likely to have many important functions in the kidney.

Salt-sensitive transcriptome of isolated kidney distal tubule cells.

In the distal kidney tubule, the steroid hormone aldosterone regulates sodium reabsorption via the epithelial sodium channel (ENaC). Most studies seeking to identify ENaC-regulating aldosterone-induced proteins have used transcriptional profiling of cultured cells. To identify salt-sensitive transcripts in an in vivo model, we used low-NaCl or high-NaCl diet to stimulate or suppress endogenous aldosterone, in combination with magnetic- and fluorescence-activated cell sorting to isolate distal tubule cells from mouse kidney for transcriptional profiling. Of the differentially expressed transcripts, 162 were more abundant in distal tubule cells isolated from mice fed low-NaCl diet, and 161 were more abundant in distal tubule cells isolated from mice fed high-NaCl diet. Enrichment analysis of Gene Ontology biological process terms identified multiple statistically overrepresented pathways among the differentially expressed transcripts that were more abundant in distal tubule cells isolated from mice fed low-NaCl diet, including ion transmembrane transport, regulation of growth, and negative regulation of apoptosis. Analysis of Gene Ontology molecular function terms identified differentially expressed transcription factors, transmembrane transporters, kinases, and G protein-coupled receptors. Finally, comparison with a recently published study of gene expression changes in distal tubule cells in response to administration of aldosterone identified 18 differentially expressed genes in common between the two experiments. When expression of these genes was measured in cortical collecting ducts microdissected from mice fed low-NaCl or high-NaCl diet, eight were differentially expressed. These genes are likely to be regulated directly by aldosterone and may provide insight into aldosterone signaling to ENaC in the distal tubule.

Renal COP9 Signalosome Deficiency Alters CUL3-KLHL3-WNK Signaling Pathway.

BACKGROUND: The familial hyperkalemic hypertension (FHHt) cullin 3 (CUL3) mutant does not degrade WNK kinases normally, thereby leading to thiazide-sensitive Na-Cl cotransporter (NCC) activation. CUL3 mutant (CUL3Δ9) does not bind normally to the COP9 signalosome (CSN), a deneddylase involved in regulating cullin-RING ligases. CUL3Δ9 also caused increased degradation of the CUL3-WNK substrate adaptor kelch-like 3 (KLHL3). Here, we sought to determine how defective CSN action contributes to the CUL3Δ9 phenotype. METHODS: The Pax8/LC1 mouse system was used to generate mice in which the catalytically active CSN subunit, Jab1, was deleted only along the nephron, after full development (KS-Jab1-/-). RESULTS: Western blot analysis demonstrated that Jab1 deletion increased the abundance of neddylated CUL3. Moreover, total CUL3 expression was reduced, suggesting decreased CUL3 stability. KLHL3 was almost completely absent in KS-Jab1-/- mice. Conversely, the protein abundances of WNK1, WNK4, and SPAK kinases were substantially higher. Activation of WNK4, SPAK, and OSR1 was indicated by higher phosphorylated protein levels and translocation of the proteins into puncta, as observed by immunofluorescence. The ratio of phosphorylated NCC to total NCC was also higher. Surprisingly, NCC protein abundance was low, likely contributing to hypokalemia and Na+ and K+ wasting. Additionally, long-term Jab1 deletion resulted in kidney damage. CONCLUSIONS: Together, the results indicate that deficient CSN binding contributes importantly to the FHHt phenotype. Although defective CUL3Δ9-faciliated WNK4 degradation likely contributes, dominant effects on KLHL3 may be a second factor that is necessary for the phenotype.

Role of adenosine signaling in coordinating cardiomyocyte function and coronary vascular growth in chronic fetal anemia.

Fetal anemia causes rapid and profound changes in cardiac structure and function, stimulating proliferation of the cardiac myocytes, expansion of the coronary vascular tree, and impairing early contraction and relaxation. Although hypoxia-inducible factor-1α is sure to play a role, adenosine, a metabolic byproduct that increases coronary flow and growth, is implicated as a major stimulus for these adaptations. We hypothesized that genes involved in myocardial adenosine signaling would be upregulated in chronically anemic fetuses and that calcium-handling genes would be downregulated. After sterile surgical instrumentation under anesthesia, gestationally timed fetal sheep were made anemic by isovolumetric hemorrhage for 1 wk (16% vs. 35% hematocrit). At 87% of gestation, necropsy was performed to collect heart tissue for PCR and immunohistochemical analysis. Anemia increased mRNA expression levels of adenosine receptors ADORA 1, ADORA2A, and ADORA2B in the left and right ventricles (adenosine receptor ADORA3 was unchanged). In both ventricles, anemia also increased expression of ectonucleoside triphosphate diphosphohydrolase 1 and ecto-5'-nucleotidase. The genes for both equilibrative nucleoside transporters 1 and 2 were expressed more abundantly in the anemic right ventricle but were not different in the left ventricle. Neither adenosine deaminase nor adenosine kinase cardiac levels were significantly changed by chronic fetal anemia. Chronic fetal anemia did not significantly change cardiac mRNA expression levels of the voltage-dependent L-type calcium channel, ryanodine receptor 1, sodium-calcium exchanger, sarcoplasmic/endoplasmic reticulum calcium transporting ATPase 2, phospholamban, or cardiac calsequestrin. These data support local metabolic integration of vascular and myocyte function through adenosine signaling in the anemic fetal heart.

Endothelial transcriptomics reveals activation of fibrosis-related pathways in hypertension.

Hypertension poses a significant challenge to vasculature homeostasis and stands as the most common cardiovascular disease in the world. Its effects are especially profound on endothelial cells that form the inner lining of the vasculature and are directly exposed to the effects of excess pressure. Here, we characterize the in vivo transcriptomic response of cardiac endothelial cells to hypertension by rapidly isolating these cells from the spontaneous hypertension mouse model BPH/2J and its normotensive BPN/3J control strain and performing and RNA sequencing on both. Comparison of transcriptional differences between these groups reveals statistically significant changes in cellular pathways consistent with cardiac fibrosis found in hypertensive animals. Importantly, many of the fibrosis-linked genes identified also differ significantly between juvenile prehypertensive and adult hypertensive BPH/2J mice, suggesting that these transcriptional differences are hypertension related. We examined the dynamic nature of these transcriptional changes by testing whether blood pressure normalization using either a calcium channel blocker (amlodipine) or a angiotensin II receptor blocker (losartan) is able to reverse these expression patterns associated with hypertension. We find that blood pressure reduction is capable of reversing some gene-expression patterns, while other transcripts are recalcitrant to therapeutic intervention. This illuminates the possibility that unmanaged hypertension may irreversibly alter some endothelial transcriptional patterns despite later intervention. This study quantifies how endothelial cells are remodeled at the molecular level in cardiovascular pathology and advances our understanding of the transcriptional events associated with endothelial response to hypertensive challenge.

The START App: a web-based RNAseq analysis and visualization resource.

Summary: Transcriptional profiling using RNA sequencing (RNAseq) has emerged as a powerful methodology to quantify global gene expression patterns in various contexts from single cells to whole tissues. The tremendous amount of data generated by this profiling technology presents a daunting challenge in terms of effectively visualizing and interpreting results. Convenient and intuitive data interfaces are critical for researchers to easily upload, analyze and visualize their RNAseq data. We designed the START (Shiny Transcriptome Analysis Resource Tool) App with these requirements in mind. This application has the power and flexibility to be resident on a local computer or serve as a web-based environment, enabling easy sharing of data between researchers and collaborators. Availability and Implementation: Source Code for the START App is written entirely in R and can be freely available to download at https://github.com/jminnier/STARTapp with the code licensed under GPLv3. It can be launched on any system that has R installed. The START App is also hosted on https://kcvi.shinyapps.io/START for researchers to temporarily upload their data. Contact: minnier@ohsu.edu

Functional screening for G protein-coupled receptor targets of 14,15-epoxyeicosatrienoic acid.

Epoxyeicosatrienoic acids (EETs) are potent vasodilators that play important roles in cardiovascular physiology and disease, yet the molecular mechanisms underlying the biological actions of EETs are not fully understood. Multiple lines of evidence suggest that the actions of EETs are in part mediated via G protein-coupled receptor (GPCR) signaling, but the identity of such a receptor has remained elusive. We sought to identify 14,15-EET-responsive GPCRs. A set of 105 clones were expressed in Xenopus oocyte and screened for their ability to activate cAMP-dependent chloride current. Several receptors responded to micromolar concentrations of 14,15-EET, with the top five being prostaglandin receptor subtypes (PTGER2, PTGER4, PTGFR, PTGDR, PTGER3IV). Overall, our results indicate that multiple low-affinity 14,15-EET GPCRs are capable of increasing cAMP levels following 14,15-EET stimulation, highlighting the potential for cross-talk between prostanoid and other ecosanoid GPCRs. Our data also indicate that none of the 105 GPCRs screened met our criteria for a high-affinity receptor for 14,15-EET.

Disrupting Dimerization Translocates Soluble Epoxide Hydrolase to Peroxisomes.

The epoxyeicosatrienoic acid (EET) neutralizing enzyme soluble epoxide hydrolase (sEH) is a neuronal enzyme, which has been localized in both the cytosol and peroxisomes. The molecular basis for its dual localization remains unclear as sEH contains a functional peroxisomal targeting sequence (PTS). Recently, a missense polymorphism was identified in human sEH (R287Q) that enhances its peroxisomal localization. This same polymorphism has also been shown to generate weaker sEH homo-dimers. Taken together, these observations suggest that dimerization may mask the sEH PTS and prevent peroxisome translocation. In the current study, we test the hypothesis that dimerization is a key regulator of sEH subcellular localization. Specifically, we altered the dimerization state of sEH by introducing substitutions in amino acids responsible for the dimer-stabilizing salt-bridge. Green Fluorescent Protein (GFP) fusions of each of mutants were co-transfected into mouse primary cultured cortical neurons together with a PTS-linked red fluorescent protein to constitutively label peroxisomes. Labeled neurons were analyzed using confocal microscopy and co-localization of sEH with peroxisomes was quantified using Pearson's correlation coefficient. We find that dimer-competent sEH constructs preferentially localize to the cytosol, whereas constructs with weakened or disrupted dimerization were preferentially targeted to peroxisomes. We conclude that the sEH dimerization status is a key regulator of its peroxisomal localization.