Vasodilatory effect of pentoxifylline in isolated equine digital veins

The direct vasodilatory action of pentoxifylline (1-(5-oxohexyl)-3,7-dimethylxanthine) and its signalling pathway was evaluated in equine digital veins. Cumulative concentration-response curves to pentoxifylline (1 nM to 300 μM) were recorded in phenylephrine-precontracted equine digital vein rings under different experimental conditions. Relaxation to pentoxifylline was partially inhibited by endothelium removal, but was unaltered by CGS-15943 (a non-xanthine adenosine receptor antagonist; 3 μM). Nitric oxide synthase (NOS), soluble guanylate cyclase and cyclooxygenase (COX) inhibitors (Nω-nitro-L-arginine methyl ester (100 μM), ODQ (30 μM) and indomethacin (10 μM), respectively) significantly reduced the maximum relaxation induced by pentoxifylline. Moreover, pentoxifylline-induced relaxation was strongly reduced by Rp-8-Br-PET-cyclic guanosine monophosphate-S (a protein kinase G inhibitor; 3 μM), but remained unaffected by H-89 (a protein kinase A inhibitor; 2 μM). Pentoxifylline-induced relaxation was associated with a 3.4-fold increase in tissue cGMP content. To investigate whether pentoxifylline can affect cAMP- and cGMP-mediated relaxations, curves to forskolin, to sodium nitroprusside (SNP) and 8-bromo-cGMP were also recorded in endothelium-denuded equine digital vein rings pretreated with pentoxifylline (10 and 100 μM). Pentoxifylline only potentiated the SNP-mediated relaxation at the highest concentration (100 μM). Thus, pentoxifylline relaxed equine digital veins via endothelium-dependent and endothelium-independent components. The effect was mediated through both the NOS and COX pathways and could also result from inhibition of cGMP specific-phosphodiesterase activity at the highest concentrations used

The Role of ZIP9 and Androgen Receptor in the Establishment of Tight Junctions between Adult Rat Sertoli Cells

The blood–testis barrier (BTB) is formed from tight junctions (TJs) between Sertoli cells. This dynamic structure, which establishes an immune-privileged environment protecting haploid germ cells formed in puberty from cells of the innate immune system, protects male fertility. Testosterone produced in Leydig cells is one of the main regulators of TJ protein expression and BTB dynamics. Nevertheless, although it has been assumed that testosterone effects on TJs and BTB are mediated through the classical androgen receptor (AR), newer results call the importance of this receptor into question. ZIP9, a recently identified androgen receptor of plasma membranes, mediates testosterone effects that promote the expression of TJ proteins and TJ formation in a rat Sertoli cell line that lacks the classical AR. Although these findings suggest that ZIP9 mediates these testosterone effects, participation of the classical AR in these events cannot be excluded. Here we used immortalized adult rat Sertoli cells that express both ZIP9 and AR and addressed the involvement of these receptors in the stimulation of TJ protein expression and TJ formation in response to testosterone and to the androgenic peptide IAPG that acts via ZIP9. We find that both testosterone and IAPG trigger the so-called non-classical signaling pathway of testosterone and stimulate the expression of TJ-associated proteins and TJ formation. Silencing classical AR expression had no effect on the responses, whereas silencing of ZIP9 expression completely blocked them. Our results demonstrate that ZIP9 is the sole androgen receptor involved in the regulation of TJ protein expression and TJ formation at the BTB

Long-Term Maintenance of Viable Adult Rat Sertoli Cells Able to Establish Testis Barrier Components and Function in Response to Androgens

A protocol for the isolation and long-term propagation of adult rat Sertoli cells (SCs) using conditional reprogramming (CR) was developed and the formation of tight junctions as an in vitro model for the blood testis barrier (BTB) was studied. Three pure primary SC lines were isolated successfully and maintained for several months without significant changes in expression levels of SC-typical markers such as SRY-box transcription factor 9 (SOX9), transferrin, clusterin, androgen receptor (AR), and GATA binding protein 1 (GATA1). In addition to AR expression, the tight junction proteins, zonula occludens-1 (ZO-1) and the junctional adhesion molecule-3 (JAM-3), were upregulated and the SC barrier integrity was enhanced by testosterone. Peritubular/myoid cells did not increase the tightness of the SC. The cytokines, interleukin-6 (IL-6), bone morphogenetic protein-2 (BMP2), and transforming growth factor beta-3 (TGF-β3), negatively affected the tightness of the SC barrier. We have established a protocol for the isolation and long-term propagation of highly pure primary adult rat SCs, which are able to respond to androgen treatments, to form tight junctions and to maintain the mRNA expression of SC-specific genes. By applying this new method, adult SCs can now be analyzed in more detail and might serve as an in vitro model for the study of many SC functions

Evaluation of the effect of sodium hypochlorite gel on composite bonding strength to enamel of primary teeth after salivary contamination: in vitro study [Primenenie gelya gipokhlorita natriya dlya uluchsheniya stsepleniya kompozitnogo materiala s emal'yu vremennykh zubov pri slyunnoi kontaminatsii]

OBJECTIVE: The study investigated the effect of 2%s odium hypochlorite gel application after post-etching salivary contamination on composite bonding strength to primary teeth enamel. MATERIAL AND METHODS: The sample consisted of 79 primary human teeth that were extracted no more than one month ago. The sample was randomly divided into four groups: (1) the control group (A) comprised 10 primary teeth, and composites were applied in the traditional manner without any salivary contamination; (2) the second group (B) consisted of 23 primary teeth in which salivary contamination was conducted after etching followed by re-etching and follow-up; (3) the third group (C) comprised 23 primary teeth in which saliva contamination was done after etching followed by washing, drying, and follow-up; and (4) the fourth group (D) comprised 23 primary teeth, in which salivary contamination was conducted after etching followed by application of sodium hypochlorite gel and follow-up. The samples were tested using the Testometric Tensile Strength Device (Testometric M350-10 kN, Testometric Ltd., UK) to measure the composite bonding strength to enamel of primary teeth. RESULTS: The arithmetic mean strength values in the research sample were ordered as followed: (A=13.39 MPa) > (D=11.82 MPa) > (C=8.07 MPa) > (B=6.15 MPa). The application of sodium hypochlorite gel after salivary contamination significantly improved the composite bonding strength to primary teeth enamel when compared with re-etching or only washing and drying. CONCLUSION: 15 s exposition of sodium hypochlorite gel with subsequent rinse and drying is recommended in case of saliva contamination of etched primary tooth enamel surface.ЦЕЛЬ ИССЛЕДОВАНИЯ: Изучение воздействия геля, содержащего 2% гипохлорита натрия, на последующее сцепление композитного материала и эмали после попадания слюны на поверхность эмали, которая была обработана 37% ортофосфорной кислотой TetricN-Etch (Ivoclar Vivadent). МАТЕРИАЛ И МЕТОДЫ: Исследованы 79 временных зубов, которые были удалены не более 1 мес назад. Данные зубы были поделены случайным образом на 4 группы: группа A включала 10 временных зубов, эмаль которых была обработана классическим методом без нанесения слюны, группа B — 23 временных зуба, эмаль которых была протравлена с последующим нанесением слюны и повторным протравлением, группа C — 23 временных зуба, эмаль которых была протравлена, после чего на нее нанесли слюну, рабочее поле промыли водой и высушили, а затем выполнили композитную реставрацию, группа D — 23 временных зуба, эмаль которых была обработана слюной после протравливания, затем был использован гель гипохлорита натрия, и работа была продолжена. Исследование проводилось на аппарате для определения прочности при разрыве (Testometric M350-10 kN, «Testometric Ltd.», Великобритания). РЕЗУЛЬТАТЫ: Среднеарифметические значения сцепления композита и эмали зубов распределились следующим образом: (A=13,39 МПа) > (D=11,82 МПа) > (C=8,07 МПа) > (B=6,15 МПа). Исследования показали, что использование гипохлорита натрия в виде геля после попадания слюны на поверхность обработанной эмали укрепляет сцепление композита с эмалью временных зубов статистически значимо больше, чем повторное протравливание или промывание водой с высушиванием. ЗАКЛЮЧЕНИЕ: Во время работы с композитными материалами при попадании слюны после стадии протравливания на рабочее поле эмали временного зуба авторы рекомендуют наносить гель гипохлорита натрия в течение 15 с с последующим промыванием и высушиванием

The Effects of Tetrapeptides Designed to Fit the Androgen Binding Site of ZIP9 on Myogenic and Osteogenic Cells

ZIP9 is a recently identified membrane-bound androgen receptor of physiological significance that may mediate certain physiological responses to androgens. Using in silico methods, six tetrapeptides with the best docking properties at the testosterone binding site of ZIP9 were synthesized and further investigated. All tetrapeptides displaced T-BSA-FITC, a membrane-impermeable testosterone analog, from the surface of mouse myogenic L6 cells that express ZIP9 but not the classical androgen receptor (AR). Silencing the expression of ZIP9 with siRNA prevented this labeling. All tetrapeptides were found to be pro-androgenic; in L6 cells they stimulated the expression of myogenin, triggered activation of focal adhesion kinase, and prompted the fusion of L6 myocytes to syncytial myotubes. In human osteoblastic SAOS-2 cells that express AR and ZIP9, they reduced the expression of alkaline phosphatase and stimulated mineralization. These latter effects were prevented by silencing ZIP9 expression, indicating that the osteoblast/osteocyte conversion is exclusively mediated through ZIP9. Our results demonstrate that the synthetic tetrapeptides, by acting as ZIP9-specific androgens, have the potential to replace testosterone or testosterone analogs in the treatment of bone- or muscle-related disorders by circumventing the undesirable effects mediated through the classical AR