Jessica R. Hughes

P2Y Receptor Modulation of ATP Release in the Urothelium

The release of ATP from the urothelium in response to stretch during filling demonstrates the importance of the purinergic system for the physiological functioning of the bladder. This study examined the effect of P2 receptor agonists on ATP release from two urothelial cell lines (RT4 and UROtsa cells). Hypotonic Krebs was used as a stretch stimulus. Incubation of urothelial cells with high concentrations of the P2Y agonist ADP induced ATP release to a level that was 40-fold greater than hypotonic-stimulated ATP release (P < 0.0011, ADP EC50 1.8 µM). Similarly, an increase in ATP release was also observed with the P2Y agonist, UTP, up to a maximum of 70% of the hypotonic response (EC50 0.62 µM). Selective P2 receptor agonists, αβ-methylene-ATP, ATP-γ-S, and 2-methylthio-ADP had minimal effects on ATP release. ADP-stimulated ATP release was significantly inhibited by suramin (100 µM, P = 0.002). RT4 urothelial cells break down nucleotides (100 µM) including ATP, ADP, and UTP to liberate phosphate. Phosphate liberation was also demonstrated from endogenous nucleotides with approximately 10% of the released ATP broken down during the incubation. These studies demonstrate a role for P2Y receptor activation in stimulation of ATP release and emphasize the complexity of urothelial P2 receptor signalling.

Characterisation of sphingolipids in the human lens by thin layer chromatography-desorption electrospray ionisation mass spectrometry.

The lipidome of the human lens is unique in that cholesterol and dihydrosphingomyelin are the dominant classes. Moreover, the lens lipidome is not static with dramatic changes in several sphingolipid classes associated with both aging and cataract. Accordingly, there is a clear need to expand knowledge of the molecular species that constitute the human lens sphingolipidome. In this study, human lens lipids have been extracted and separated by thin-layer chromatography (TLC). Direct analysis of the TLC plates by desorption electrospray ionisation-mass spectrometry (DESI-MS) allowed the detection over 30 species from 11 classes of sphingolipids. Significantly, novel classes of lens lipids including sulfatides, dihydrosulfatides, lactosylceramide sulfates and dihydrolactosylceramide sulfates were identified.

Effect of Inflammatory Mediators on ATP Release of Human Urothelial RT4 Cells

Inflammation is an important contributor to the aetiology of a number of bladder dysfunctions including interstitial cystitis, painful bladder syndrome, and overactive bladder. The aim of this study was to examine the effects of inflammatory mediators on urothelial ATP release. Human urothelial RT4 cells were exposed to normal buffer or varying concentrations of inflammatory mediators (bradykinin, histamine, and serotonin) in the presence or absence of hypotonic stretch stimuli (1 : 2 dilution of Krebs-Henseleit buffer). Others have demonstrated that bradykinin increased stretch-induced ATP release; however, we observed no change in control or stretch-induced ATP release with bradykinin. Pretreatment of RT4 cells with histamine or serotonin decreased stretch-induced ATP release (P = 0.037, P = 0.040, resp.). Previous studies have demonstrated increased ATP release in response to inflammation utilising whole bladder preparations in contrast to our simple model of cultured urothelial cells. The current study suggests that it is unlikely that there is a direct interaction between the release of inflammatory mediators and increased ATP release, but rather more complex interactions occurring in response to inflammation that lead to increased bladder sensation.

No turnover in lens lipids for the entire human lifespan

Lipids are critical to cellular function and it is generally accepted that lipid turnover is rapid and dysregulation in turnover results in disease (Dawidowicz 1987; Phillips et al., 2009; Liu et al., 2013). In this study, we present an intriguing counter-example by demonstrating that in the center of the human ocular lens, there is no lipid turnover in fiber cells during the entire human lifespan. This discovery, combined with prior demonstration of pronounced changes in the lens lipid composition over a lifetime (Hughes et al., 2012), suggests that some lipid classes break down in the body over several decades, whereas others are stable. Such substantial changes in lens cell membranes may play a role in the genesis of age-related eye disorders. Whether long-lived lipids are present in other tissues is not yet known, but this may prove to be important in understanding the development of age-related diseases. DOI: http://dx.doi.org/10.7554/eLife.06003.001

Unsuccessful ageing: dramatic changes to the human lens lipidome with age

Purpose: In peripheral vision, the natural pupil is elliptical and Zernike circular aberration polynomials cannot be directly used to describe the associated wavefront. We compare two methods for solving this problem. Methods: The stretched elliptical approach uses an elliptical pupil matching actual pupil shape. This is stretched along its minor axis to become a circle, allowing circular polynomials to be used. Another approach involves circular pupils with diameter matching either the larger or smaller axis of the elliptical pupil, again with circular polynomials. We investigated advantages and disadvantages of these alternatives using real and model eye data. Results: For 5-mm entrance pupils, the approaches give similar aberration coefficients to 20° from fixation: beyond this, they depart considerably. Advantages of the stretched elliptical relative to the circular pupil approach include: more physiological; aberrations include all information from the true pupil (may not be true of circular pupils smaller than the elliptical pupil); root-mean-squared aberrations are given directly from aberration coefficients; it is compatible with optical design programs. Its disadvantages include: greater mathematical complication; distortion of wavefront shape by stretching; comparing coefficients for different angles may not be valid; and MTFs and PSFs are distorted if derived directly from wave aberration coefficients. There are ways to compensate for some of the disadvantages of each of the methods. Conclusions: In spite of theoretical merits of the stretched elliptical pupil approach, the simplicity of the circular pupil approach and the ease with which its results can be understood by clinicians may make it preferable. AOVSM Free Papers Abstracts