VNUT and VMAT2 segregate within sympathetic varicosities and localize near preferred Cav2 isoforms in the rat tail artery.

ATP and norepinephrine (NE) are coreleased from peripheral sympathetic nerve terminals. Whether they are stored in the same vesicles has been debated for decades. Preferential dependence of NE or ATP release on Ca2+ influx through specific voltage-gated Ca2+ channel (Cav2) isoforms suggests that NE and ATP are stored in separate vesicle pools, but simultaneous imaging of NE and ATP containing vesicles within single varicosities has not been reported. We conducted an immunohistochemical study of vesicular monoamine transporter 2 (VMAT2/SLC18A2) and vesicular nucleotide translocase (VNUT/SLC17A9) as markers of vesicles containing NE and ATP in sympathetic nerves of the rat tail artery. A large fraction of varicosities exhibited neighboring, rather than overlapping, VNUT and VMAT2 fluorescent puncta. VMAT2, but not VNUT, colocalized with synaptotagmin 1. Cav2.1, Cav2.2, and Cav2.3 are expressed in nerves in the tunica adventitia. VMAT2 preferentially localized adjacent to Cav2.2 and Cav2.3 rather than Cav2.1. VNUT preferentially localized adjacent to Cav2.3 > Cav2.2 >> Cav2.1. With the use of wire myography, inhibition of field-stimulated vasoconstriction with the Cav2.3 blocker SNX-482 (0.25 µM) mimicked the effects of the P2X inhibitor suramin (100 µM) rather than the α-adrenergic inhibitor phentolamine (10 µM). Variable sensitivity to SNX-482 and suramin between animals closely correlated with Cav2.3 staining. We concluded that a majority of ATP and NE stores localize to separate vesicle pools that use different synaptotagmin isoforms and that localize near different Cav2 isoforms to mediate vesicle release. Cav2.3 appears to play a previously unrecognized role in mediating ATP release in the rat tail artery. NEW & NOTEWORTHY Immunofluorescence imaging of vesicular nucleotide translocase and vesicular monoamine transporter 2 in rat tail arteries revealed that ATP and norepinephrine, classical cotransmitters, localize to well-segregated vesicle pools. Furthermore, vesicular nucleotide translocase and vesicular monoamine transporter 2 exhibit preferential localization with specific Cav2 isoforms. These novel observations address long-standing debates regarding the mechanism(s) of sympathetic neurotransmitter corelease.

Droplet digital PCR shows the D-Loop to be an error prone locus for mitochondrial DNA copy number determination.

Absolute quantification of mitochondrial DNA copy number (mCN) provides important insights in many fields of research including cancer, cardiovascular and reproductive health. Droplet digital PCR (ddPCR) natively reports absolute copy number, and we have developed a single-dye, multiplex assay to measure rat mCN that is accurate, precise and affordable. We demonstrate simple methods to optimize this assay and to determine nuclear reference pseudogene copy number to extend the range of mCN that can be measured with this assay. We evaluated two commonly used mitochondrial DNA reference loci to determine mCN, the ND1 gene and the D-Loop. Harnessing the absolute measures of ddPCR, we found that the D-Loop amplifies with a copy number of ~1.0-1.5 relative to other sites on the mitochondrial genome. This anomalous copy number varied significantly between rats and tissues (aorta, brain, heart, liver, soleus muscle). We advocate for avoiding the D-Loop as a mitochondrial reference in future studies of mCN. Further, we report a novel approach to quantifying immunolabelled mitochondrial DNA that provides single-cell estimates of mCN that closely agree with the population analyses by ddPCR. The combination of these assays represents a cost-effective and powerful suite of tools to study mCN.

pH Modulation of Voltage-Gated Sodium Channels.

Changes in blood and tissue pH accompany physiological and pathophysiological conditions including exercise, cardiac ischemia, ischemic stroke, and cocaine ingestion. These conditions are known to trigger the symptoms of electrical diseases in patients carrying sodium channel mutations. Protons cause a diverse set of changes to sodium channel gating, which generally lead to decreases in the amplitude of the transient sodium current and increases in the fraction of non-inactivating channels that pass persistent currents. These effects are shared with disease-causing mutants in neuronal, skeletal muscle, and cardiac tissue and may be compounded in mutants that impart greater proton sensitivity to sodium channels, suggesting a role of protons in triggering acute symptoms of electrical disease.In this chapter, we review the mechanisms of proton block of the sodium channel pore and a suggested mode of action by which protons alter channel gating. We discuss the available data on isoform specificity of proton effects and tissue level effects. Finally, we review the role that protons play in disease and our own recent studies on proton-sensitizing mutants in cardiac and skeletal muscle sodium channels.

Compound heterozygous TRPV4 mutations in two siblings with a complex phenotype including severe intellectual disability and neuropathy.

TRPV4 encodes a polymodal calcium-permeable plasma membrane channel. Dominant pathogenic mutations in TRPV4 lead to a wide spectrum of abnormal phenotypes. This is the first report of biallelic TRPV4 mutations and we describe two compound heterozygous siblings presenting with a complex phenotype including severe neuromuscular involvement. In light of previously well described dominant inheritance for TRPV4-related neuromuscular disease, our study suggests a role for compound heterozygosity and loss-of-function as a potential novel disease mechanism for this group of disorders. Profound intellectual disability was also noted in both affected children, suggesting that TRPV4 may be necessary for normal brain development.

Colocalization of voltage-gated Na+ channels with the Na+/Ca2+ exchanger in rabbit cardiomyocytes during development.

Reverse-mode activity of the Na(+)/Ca(2+) exchanger (NCX) has been previously shown to play a prominent role in excitation-contraction coupling in the neonatal rabbit heart, where we have proposed that a restricted subsarcolemmal domain allows a Na(+) current to cause an elevation in the Na(+) concentration sufficiently large to bring Ca(2+) into the myocyte through reverse-mode NCX. In the present study, we tested the hypothesis that there is an overlapping expression and distribution of voltage-gated Na(+) (Na(v)) channel isoforms and the NCX in the neonatal heart. For this purpose, Western blot analysis, immunocytochemistry, confocal microscopy, and image analyses were used. Here, we report the robust expression of skeletal Na(v)1.4 and cardiac Na(v)1.5 in neonatal myocytes. Both isoforms colocalized with the NCX, and Na(v)1.5-NCX colocalization was not statistically different from Na(v)1.4-NCX colocalization in the neonatal group. Western blot analysis also showed that Na(v)1.4 expression decreased by sixfold in the adult (P < 0.01) and Na(v)1.1 expression decreased by ninefold (P < 0.01), whereas Na(v)1.5 expression did not change. Although Na(v)1.4 underwent large changes in expression levels, the Na(v)1.4-NCX colocalization relationship did not change with age. In contrast, Na(v)1.5-NCX colocalization decreased ∼50% with development. Distance analysis indicated that the decrease in Na(v)1.5-NCX colocalization occurs due to a statistically significant increase in separation distances between Na(v)1.5 and NCX objects. Taken together, the robust expression of both Na(v)1.4 and Na(v)1.5 isoforms and their colocalization with the NCX in the neonatal heart provides structural support for Na(+) current-induced Ca(2+) entry through reverse-mode NCX. In contrast, this mechanism is likely less efficient in the adult heart because the expression of Na(v)1.4 and NCX is lower and the separation distance between Na(v)1.5 and NCX is larger.

Angiotensin-converting enzyme (ACE) inhibitors modulate cellular retinol-binding protein 1 and adiponectin expression in adipocytes via the ACE-dependent signaling cascade.

Inhibitors of the angiotensin-converting enzyme (ACE) decrease angiotensin II production and activate an intracellular signaling cascade that affects gene expression in endothelial cells. Because ACE inhibitors have been reported to delay the onset of type 2 diabetes, we determined ACE signaling-modulated gene expression in endothelial cells and adipocytes. Using differential gene expression analysis, several genes were identified that were 3-fold up- or down-regulated by ramiprilat in cells expressing wild-type ACE versus cells expressing a signaling-dead ACE mutant. One up-regulated gene was the cellular retinol-binding protein 1 (CRBP1). In adipocytes, the overexpression of CRBP1 enhanced (4- to 5-fold) the activity of promoters containing response elements for retinol-dependent nuclear receptors [retinoic acid receptor (RAR) and retinoid X receptor (RXR)] or peroxisome proliferator-activated receptors (PPAR). CRBP1 overexpression also enhanced the promoter activity (by 470 +/- 40%) and expression/release of the anti-inflammatory and antiatherogenic adipokine adiponectin (cellular adiponectin by 196 +/- 24%, soluble adiponectin by 228 +/- 74%). Significantly increased adiponectin secretion was also observed after ACE inhibitor treatment of human preadipocytes, an effect prevented by small interfering RNA against CRBP1. Furthermore, in ob/ob mice, ramipril markedly potentiated both the basal (approximately 2-fold) and rosiglitazonestimulated circulating levels of adiponectin. In patients with coronary artery disease or type 2 diabetes, ACE inhibition also significantly increased plasma adiponectin levels (1.6- or 2.1-fold, respectively). In summary, ACE inhibitors affect adipocyte homeostasis via CRBP1 through the activation of RAR/RXR-PPAR signaling and up-regulation of adiponectin. The latter may contribute to the beneficial effects of ACE inhibitors on the development of type 2 diabetes in patients with an activated renin-angiotensin system.

Angiotensin-converting enzyme (ACE) dimerization is the initial step in the ACE inhibitor-induced ACE signaling cascade in endothelial cells.

The binding of angiotensin-converting enzyme (ACE) inhibitors to ACE initiates a signaling cascade that involves the phosphorylation of the enzyme on Ser1270 as well as activation of the c-Jun NH2-terminal kinase (JNK) and leads to alterations in gene expression. To clarify how ACE inhibitors activate this pathway, we determined their effect on the ability of the enzyme to dimerize and the role of ACE dimerization in the initiation of the ACE signaling cascade. In endothelial cells, ACE was detected as a monomer as well as a dimer in native gel electrophoresis and dimerization/oligomerization was confirmed using the split-ubiquitin assay in yeast. ACE inhibitors elicited a rapid, concentration-dependent increase in the dimer/monomer ratio that correlated with that of the ACE inhibitorinduced phosphorylation of ACE. Cell treatment with galactose and glucose to prevent the putative lectin-mediated self-association of ACE or with specific antibodies shielding the N terminus of ACE failed to affect either the basal or the ACE inhibitor-induced dimerization of the enzyme. In ACE-expressing Chinese hamster ovary cells, ACE inhibitors elicited ACE dimerization and phosphorylation as well as the activation of JNK with similar kinetics to those observed in endothelial cells. However, these effects were prevented by the mutation of the essential Zn2+-complexing histidines in the C-terminal active site of the enzyme. Mutation of the N-terminal active site of ACE was without effect. Together, our data suggest that ACE inhibitors can initiate the ACE signaling pathway by inducing ACE dimerization, most probably via the C-terminal active site of the enzyme.

Ragaglitazar: a novel PPAR alpha PPAR gamma agonist with potent lipid-lowering and insulin-sensitizing efficacy in animal models.

Ragaglitazar [(-) DRF 2725; NNC 61-0029] is a coligand of PPARalpha and PPARgamma. In ob/ob mice, ragaglitazar showed significant reduction in plasma glucose, triglyceride and insulin (ED50 values <0.03, 6.1 and <0.1 mg kg-1). These effects are three-fold better than rosiglitazone and KRP-297. In Zucker fa/fa rats, ragaglitazar showed dose-dependent reduction in triglyceride and insulin, hepatic triglyceride secretion and triglyceride clearance kinetics (maximum of 74, 53, 32 and 50% at 3 mg kg-1), which are better than rosiglitazone and KRP-297. In a high-fat-fed hyperlipidaemic rat model, the compound showed an ED50 of 3.95, 3.78 mg kg-1 for triglyceride and cholesterol lowering, and 0.29 mg kg-1 for HDL-C increase. It also showed improvement in clearance of plasma triglyceride and hepatic triglyceride secretion rate. All these effects are 3-10-fold better than fenofibrate and KRP-297. Ragaglitazar treatment showed significant reduction in plasma Apo B and Apo CIII levels, and increase in liver CPT1 and CAT activity and ACO mRNA. Significant increase of both liver and fat LPL activity and fat aP2 mRNA was also observed. In a high-fat-fed hamster model, ragaglitazar at 1 mg kg-1 showed 83 and 61% reduction in triglyceride and total cholesterol, and also 17% reduction in fat feed-induced body weight increase. In these hyperlipidaemic animal models, PPARgamma ligands failed to show any significant efficacy. Taken together, ragaglitazar shows better insulin-sensitizing and lipid-lowering potential, as compared to the standard compounds.

PAT5A: a partial agonist of peroxisome proliferator-activated receptor gamma is a potent antidiabetic thiazolidinedione yet weakly adipogenic.

PAT5A [5-[4-[N-(2-pyridyl)-(2S)-pyrrolidine-2-methoxyl]phenylmethylene[thiazolidine-2,4-dione, malic acid salt]], a chemically distinct unsaturated thiazolidinedione, activates peroxisome proliferator-activated receptor gamma (PPARgamma) submaximally in vitro with the binding affinity approximately 10 times less than that of rosiglitazone, a highly potent thiazolidinedione. PAT5A reduces plasma glucose level and improves insulin sensitivity in insulin resistant db/db mice, similar to that of rosiglitazone, while exerting a relatively weak adipogenic effect. In contrast to rosiglitazone, PAT5A inhibits cholesterol and fatty acid biosynthesis suggesting that PAT5A possesses a unique receptor-independent non-PPAR related property. PAT5A induces qualitatively similar but quantitatively different protease digestion patterns and interacts with PPARgamma differently than rosiglitazone. PAT5A shows differential cofactor recruitment and gene activation than that of rosiglitazone. Thus, the partial agonism of PAT5A to PPARgamma together with its receptor independent effects may contribute to its antidiabetic potency similar to rosiglitazone in vivo despite reduced affinity for PPARgamma. These biological effects suggest that PAT5A is a PPARgamma modulator that activates some (insulin sensitization), but not all (adipogenesis), PPARgamma-signaling pathways.

DRF 2655: a unique molecule that reduces body weight and ameliorates metabolic abnormalities.

OBJECTIVE: Preclinical evaluation of DRF 2655, a peroxisome proliferator-activated receptor alpha (PPARalpha) and PPARgamma agonist, as a body-weight lowering, hypolipidemic and euglycemic agent. RESEARCH METHODS AND PROCEDURES: DRF 2655 was studied in different genetic, normal, and hyperlipidemic animal models. HEK 293 cells were used to conduct the reporter-based transactivation of PPARalpha and PPARgamma. To understand the biochemical mechanism of lipid-, body-weight-, and glucose-lowering effects, activities of key beta-oxidation and lipid catabolism enzymes and gluconeogenic enzymes were studied in db/db mice treated with DRF 2655. 3T3L1 cells were used for adipogenesis study, and HepG2 cells were used to study the effect of DRF 2655 on total cholesterol and triglyceride synthesis using [(14)C]acetate and [(3)H]glycerol. RESULTS: DRF 2655 showed concentration-dependent transactivation of PPARalpha and PPARgamma. In the 3T3L1 cell-differentiation study, DRF 2655 and rosiglitazone showed 369% and 471% increases, respectively, in triglyceride accumulation. DRF 2655 showed body-weight lowering and euglycemic and hypolipidemic effects in various animal models. db/db mice treated with DRF 2655 showed 5- and 3.6-fold inhibition in phosphoenolpyruvate carboxykinase and glucose 6-phosphatase activity and 651% and 77% increases in the beta-oxidation enzymes carnitine palmitoyltransferase and carnitine acetyltransferase, respectively. HepG2 cells treated with DRF 2655 showed significant reduction in lipid synthesis. DISCUSSION: DRF 2655 showed excellent euglycemic and hypolipidemic activities in different animal models. An exciting finding is its body-weight lowering effect in these models, which might be mediated by the induction of target enzymes involved in hepatic lipid catabolism through PPARalpha activation.