Hyaluronan Export through Plasma Membranes Depends on Concurrent K+ Efflux by Kir Channels

Hyaluronan is synthesized within the cytoplasm and exported into the extracellular matrix through the cell membrane of fibroblasts by the MRP5 transporter. In order to meet the law of electroneutrality, a cation is required to neutralize the emerging negative hyaluronan charges. As we previously observed an inhibiting of hyaluronan export by inhibitors of K+ channels, hyaluronan export was now analysed by simultaneously measuring membrane potential in the presence of drugs. This was done by both hyaluronan import into inside-out vesicles and by inhibition with antisense siRNA. Hyaluronan export from fibroblast was particularly inhibited by glibenclamide, ropivacain and BaCl2 which all belong to ATP-sensitive inwardly-rectifying Kir channel inhibitors. Import of hyaluronan into vesicles was activated by 150 mM KCl and this activation was abolished by ATP. siRNA for the K+ channels Kir3.4 and Kir6.2 inhibited hyaluronan export. Collectively, these results indicated that hyaluronan export depends on concurrent K+ efflux

Influence of progesterone, hyaluronan and growth hormone on ciliary transport and secretion in the bovine oviduct

Die frühe embryo-maternale Kommunikation findet bereits im Eileiter statt und bildet eine essentielle Voraussetzung für eine erfolgreiche Trächtigkeit. Zur Analyse der beteiligten Signaltransduktionswege wurden bisher überwiegend Untersuchungen unter in vitro Bedingungen durchgeführt. Ziel dieser Arbeit war es daher, unter nahen in vivo Bedingungen den Einfluss von Progesteron (P), Hyaluronsäure (HA) und Wachstumshormon (GH) auf den ziliären Transport und die Sekretion im Eileiter am Modell des Rindes zu untersuchen. Zu diesem Zweck wurden ex vivo Organkulturen des Eileiters mit einem neu etablierten digitalen Videomikroskop untersucht. Dabei wurden mit Hilfe der Software StreamPix und Image-Pro Plus die Partikeltransportgeschwindigkeit (PTG) und die Zilienschlagfrequenz (ZSF) analysiert. Zur Ermittlung der ZSF wurde zusätzlich eine Fast Fourier Transformation durchgeführt. Zudem erfolgten histochemische und immunhistochemische Analysen. Während des Östrus beträgt die basale PTG in der Ampulla 234,7 ± 18,9 µm/s (n = 4) und die ZSF 17,8 ± 0,6 Hz (n = 10). HA bewirkt in der Ampulla nach 15 min. einen signifikanten Anstieg der ZSF um 25,9 % (n = 7) und könnte damit als natürlicher Bestandteil der Eileiterflüssigkeit und des COCs eine wichtige Rolle beim Oozyten-Pickup spielen sowie an dem physiologischen Anstieg der ZSF nach der Ovulation beteiligt sein, um den Flüssigkeitsstrom der viskösen Follikelflüssigkeit aufrecht zu erhalten. Zudem löst exogen zugefügte HA im Metöstrus einen signifikanten Anstieg (n =6) der Synthese von sauren Mukopolysacchariden in den sekretorischen Zellen der Ampulla aus und trägt damit zu einer erfolgreichen Gametogenese sowie Fertilisation bei. Progesteron senkt die ZSF in der Ampulla signifikant um 21,6 % (n = 6). Eine Begründung dafür könnte die untergeordnete Rolle der ziliären Aktivität im P dominierenden Diöstrus darstellen, da in diesem Zyklusabschnitt kein Transport der Eizelle bzw. des Embryos stattfindet. Dies spiegelt sich auch in der signifikant reduzierten Synthese von sauren Mukopolysacchariden wider (n = 7), da auch eine nutritive Versorgung im Diöstrus nicht erforderlich ist. In der frühen Trächtigkeit ist die Bereitstellung von Nährstoffen hingegen von großer Bedeutung, wodurch sich die starke Sekretion von Glykoproteinen über ausgeschleuste Vesikel in der Ampulla begründet. Die Expression des PRs ist 1,5 Tage, nachdem der Embryo die Ampulla verlassen hat, ipsilateral signifikant geringer (64.1 %) als kontralateral (n = 4), was auf einen lokalen Wirkungsmechanismus des Embryos hinweist. Im Uterus wird der PR bei der Ankunft des Embryos ipsi- als auch kontralateral stark exprimiert. Mit Hilfe dieser Arbeit konnte gezeigt werden, dass die ziliäre Aktivität im bovinen Eileiter sowohl endokrin (P) als auch parakrin (HA) über lokale Signale reguliert wird. Durch die gewonnenen Erkenntnisse wird zu einem besseren Verständnis der kausalen Zusammenhänge bei Sub- und Infertilitäten bezüglich eines gestörten Gameten- bzw. Embryotransportes beigetragen.The early embryo-maternal communication occurs to be in the oviduct and provides an essential prerequisite for successful pregnancy. In most instances, studies which analyze the involved signaling pathways are based on in vitro conditions. Therefore the aim of our study was to investigate the influence of progesterone (P), hyaluronan (HA) and growth hormone (GH) on ciliary transport and secretion in the bovine oviduct under near in vivo conditions. For this object we analyzed ex vivo organ cultures of the oviduct with a new established digital videomicroscope. Particletransport speed (PTS) and ciliary beating frequency (CBF) were evaluated by means of the software StreamPix and Image - Pro Plus. To determine CBF a Fast Fourier Transformation was done additionally. Moreover histochemic and immunhistochemic analyzes were performed. In the bovine estrus ampulla, basal PTS averages 234.7 ± 18.9 µm/s (n = 4) and basal CBF 17.8 ± 0.6 Hz (n = 10). HA causes a significant increase (25.9 %) of the CBF in the ampulla after an incubation of 15 minutes (n = 7). As natural component of oviductal fluid and the cumulus oocyte complex, HA could play an important role in the oocyte pickup as well as be involved in the physiological increase of ciliary beating after ovulation to ensure a continuous current of the viscous follicle fluid. Furthermore exogene HA increases the synthesis of acid mucopolysaccharides signicficantly (n = 6) in the secretory cells of the ampulla from cows in metestrus to provide successful gametogenesis and fertilization. Progesterone significantly decreases (21.6 %) CBF in the ampulla (n = 6). A potential reason could be the subordinate role of ciliary activity in the P dominant diestrus, when oocyte- and embryo transport does not occur. This is also reflected in significant reduced synthesis of acid mucopolysaccharides (n = 7) since nutritive maintenance is not necessary in diestrus. However, in the early pregnancy the supply of nutrients is very important, wherefore a strong secretion by vesicles of glycoproteins arises in the oviduct. 1.5 days after the embryo leaves the ampulla PR expression is significantly less (64.1 %) expressed in ipsilateral ampulla than in contralateral ampulla (n = 4), which indicates a local mechanism of the embryo. When the embryo enters the uterus PR is expressed very strong both ipsi- and contralateral.With the aid of this study it could be shown that ciliary activity is regulated endocrine (P) and paracrine (HA) by local signals in the bovine oviduct. The gained findings contribute to a better understanding of causal relationship in sub- and infertilities respective defective gamete and embryo transport

Recovery of ΔF508-CFTR Function by Citrate

Treatment of cystic fibrosis relies so far on expensive and sophisticated drugs. A logical approach to rescuing the defective ΔF508-CFTR protein has not yet been published. Therefore, virtual docking of ATP and CFTR activators to the open conformation of the CFTR protein was performed. A new ATP binding site outside of the two known locations was identified. It was located in the cleft between the nucleotide binding domains NBD1 and NBD2 and comprised six basic amino acids in close proximity. Citrate and isocitrate were also bound to this site. Citrate was evaluated for its action on epithelial cells with intact CFTR and defective ΔF508-CFTR. It activated hyaluronan export from human breast carcinoma cells and iodide efflux, and recovered ΔF508-CFTR from premature intracellular degradation. In conclusion, citrate is an activator for ΔF508-CFTR and increases export by defective ΔF508-CFTR into the extracellular matrix of epithelial cells

Inhibition by exogenous hyaluronan.

<p>Human skin fibroblast were labelled with [³H] GlcN and incubated in the presence of the indicated exogenous hyaluronan concentrations. After 24 hours the radioactivity incorporated into [³H] hyaluronan was determined. The error bars indicate the mean of duplicate samples.</p

Inhibition of K_ir channels by siRNA.

<p>Human fibrosarcoma cells HT1080 were reverse transfected with 30 nM siRNA for K_ir3.4, three different K_ir6.2 siRNAs, a mixture of the three K_ir6.2 siRNAs or non-sense siRNA and incubated for 24 hours in serum free Quantum medium. <b>A.</b> Specific inhibition of mRNA. The downregulation of K_ir3.4 mRNA and K_ir6.2 mRNA was detected by rtPCR. <b>B.</b> The hyaluronan concentrations were determined in the culture media. The error bars indicate the standard error of 6 determinations. *p<0.01 (ANOVA test).</p

Effect of K⁺ export inhibitors on hyaluronan export and membrane potential.

<p>Human skin fibroblasts were grown in 96 well microtiter plates with increasing concentrations of valinomycin (A), glibenclamid (B), ropivacaine (C), amiloride (D), barium chloride (E) and verapamil (F) for 24 hours. The membrane potentials (- -) and hyaluronan concentrations (__) were determined as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039096#s4" target="_blank">methods</a> section. The error bars indicate the sd of 3 determinations.</p

Effect of the K⁺ channel opener pinacidil.

<p>Human skin fibroblasts were grown in 96 well microtiter plates with increasing concentrations of pinacidil for 24 hours. The hyaluronan concentrations were determined as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039096#s4" target="_blank">methods</a> section. The error bars indicate the sd of 3 determinations.</p

Action profile of ion channel inhibitors, percentage of hyaluronan export and alteration of membrane potential at maximal inhibitory concentrations.

<p>Action profile of ion channel inhibitors, percentage of hyaluronan export and alteration of membrane potential at maximal inhibitory concentrations.</p

Model of hyaluronan synthesis and export.

<p>The hyaluronan synthase assembles hyaluronan at the inner side of the plasma membrane, the chains are exported by the ABC transporter MRP5 from fibroblasts and retained on the cell surface by CD44. Concurrent K⁺ efflux is required for maintaining electroneutrality.</p