Cyclic AMP and acidic fibroblast growth factor have opposing effects on tight and adherens junctions in microvascular endothelial cells in vitro

Endothelial adherens junctions (AJ) and tight junctions (TJ) are important determinants of vascular permeability and cell morphology. Here, we investigate their regulation, in primary human placental microvascular endothelial cell (HPMEC) cultures, by either aFGF plus heparin (ECGS) or elevated cAMP. The proliferation of HPMEC was weakly stimulated by ECGS, while cAMP was inhibitory. ECGS had little effect on transendothelial resistance (TER), but increased macromolecular permeability, whereas cAMP induced a twofold increase in TER and reduced macromolecular permeability. Ultrastructurally, ECGS-treated HPMEC exhibited an "activated" phenotype typified by proliferating cells, with poorly organized cell-cell junctions, whereas cAMP-treated cells appeared quiescent and markedly flattened with extended paracellular junctions, resembling endothelium in situ. The expression and localization of junctional molecules, F-actin, and junctional phosphotyrosine were examined by confocal microscopy and immunoblotting. Junctional molecules in ECGS-treated cells were less organized at lateral membranes than in control cells, whereas in cAMP-treated cells, they were highly localized at continuous contacts. These differences correlated with the intensity of junctional phosphotyrosine, being lowest with cAMP treatment. In the AJ of ECGS-treated and control cells, beta-catenin predominated but in cAMP-treated cells, gamma-catenin/plakoglobin was enriched. In addition, cAMP upregulated junctional expression of VE-cadherin and PECAM-1 and increased the levels of the TJ molecules occludin and ZO-1. The expression levels of junctional components, and their tyrosine phosphorylation, play an important role in dynamic regulation of endothelial cell-cell junctions.     

Oesophageal atresia: The growth gap

Poor growth is an under-recognised yet significant long-term sequelae of oesophageal atresia(OA) repair. Few studies have specifically explored the reasons for growth impairment in this complex cohort. The association between poor growth with younger age and fundoplication appears to have the strongest supportive evidence, highlighting the need for early involvement of a dietitian and speech pathologist, and consideration of optimal medical reflux management prior to referring for anti-reflux surgery. However, it remains difficult to reach conclusions regarding other factors which may negatively influence growth, due to conflicting findings, inconsistent definitions and lack of validated tool utilisation. While swallowing and feeding difficulties are particularly frequent in younger children, their relationship with growth remains unclear. It is possible that these morbidities impact on the diet of children with OA, but detailed analysis of dietary composition and quality, and its relationship with these complications and growth, has not yet been conducted. Another potential area of research in OA is the role of the microbiota in growth and nutrition. While the microbiota has been linked to growth impairment in other paediatric conditions,it is yet to be investigated in OA. Further research is needed to identify the most,important contributory factors to poor growth, the role of the intestinal microbiota, and effective interventions to maximise growth and nutritional outcomes in this cohort.     

Positive and negative allosteric modulators promote biased signaling at the calcium-sensing receptor

The calcium-sensing receptor (CaSR) is a G protein-coupled receptor whose function can be allosterically modulated in a positive or negative manner by calcimimetics or calcilytics, respectively. Indeed, the second-generation calcimimetic, cinacalcet, has proven clinically useful in the treatment of chronic kidney disease patients with secondary hyperparathyroidism but is not widely used in earlier stages of renal disease due to the potential to predispose such patients to hypocalcaemia and hyperphosphatemia. The development of a biased CaSR ligand that is more selective for specific signaling pathway(s) leading only to beneficial effects may overcome this limitation. The detection of such stimulus-bias at a G protein-coupled receptor requires investigation across multiple signaling pathways and the development of methods to quantify the effects of allosteric ligands on orthosteric ligand affinity and cooperativity at each pathway. In the current study, we determined the effects of the calcimimetics, NPS-R568 or cinacalcet, and the calcilytic, NPS-2143, on Ca(o)(2+)-mediated intracellular Ca(2+) mobilization, ERK1/2 phosphorylation, and plasma membrane ruffling in a stably transfected human embryonic kidney 293-TREx c-myc-CaSR cell line and applied a novel analytical model to quantify these modulator effects. We present quantitative evidence for the generation of stimulus bias by both positive and negative allosteric modulators of the CaSR, manifested as greater allosteric modulation of intracellular Ca(2+) mobilization relative to ERK1/2 phosphorylation, and a higher affinity of the modulators for the state of the CaSR mediating plasma membrane ruffling relative to the other two pathways. Our findings provide the first evidence that an allosteric modulator used in clinical practice exhibits stimulus bias.     

Suggestive evidence for association between L‐type voltage‐gated calcium channel (CACNA1C) gene haplotypes and bipolar disorder in Latinos: a family‐based association study

Objectives: Through recent genome‐wide association studies (GWASs), several groups have reported significant association between variants in the calcium channel, voltage‐dependent, L‐type, alpha 1C subunit (CACNA1C) and bipolar disorder (BP) in European and European‐American cohorts. We performed a family‐based association study to determine whether CACNA1C is associated with BP in the Latino population. Methods: This study included 913 individuals from 215 Latino pedigrees recruited from the USA, Mexico, Guatemala, and Costa Rica. The Illumina GoldenGate Genotyping Assay was used to genotype 58 single‐nucleotide polymorphisms (SNPs) that spanned a 602.9‐kb region encompassing the CACNA1C gene including two SNPs (rs7297582 and rs1006737) previously shown to associate with BP. Individual SNP and haplotype association analyses were performed using Family‐Based Association Test (version 2.0.3) and Haploview (version 4.2) software. Results: An eight‐locus haplotype block that included these two markers showed significant association with BP (global marker permuted p = 0.0018) in the Latino population. For individual SNPs, this sample had insufficient power (10%) to detect associations with SNPs with minor effect (odds ratio = 1.15). Conclusions: Although we were not able to replicate findings of association between individual CACNA1C SNPs rs7297582 and rs1006737 and BP, we were able to replicate the GWAS signal reported for CACNA1C through a haplotype analysis that encompassed these previously reported significant SNPs. These results provide additional evidence that CACNA1C is associated with BP and provides the first evidence that variations in this gene might play a role in the pathogenesis of this disorder in the Latino population.