Extracellular nucleotides in smooth muscle contraction.

Extracellular nucleotides and nucleosides are crucial signalling molecules, eliciting diverse biological responses in almost all organs and tissues. These molecules exert their effects by activating specific nucleotide receptors, which are finely regulated by ectonucleotidases that break down their ligands. In this comprehensive review, we aim to elucidate the relevance of extracellular nucleotides as signalling molecules in the context of smooth muscle contraction, considering the modulatory influence of ectonucleotidases on this intricate process. Specifically, we provide a detailed examination of the involvement of extracellular nucleotides in the contraction of non-vascular smooth muscles, including those found in the urinary bladder, the airways, the reproductive system, and the gastrointestinal tract. Furthermore, we present a broader overview of the role of extracellular nucleotides in vascular smooth muscle contraction.

NTPDase1 Modulates Smooth Muscle Contraction in Mice Bladder by Regulating Nucleotide Receptor Activation Distinctly in Male and Female.

Nucleotides released by smooth muscle cells (SMCs) and by innervating nerve terminals activate specific P2 receptors and modulate bladder contraction. We hypothesized that cell surface enzymes regulate SMC contraction in mice bladder by controlling the concentration of nucleotides. We showed by immunohistochemistry, enzymatic histochemistry, and biochemical activities that nucleoside triphosphate diphosphohydrolase-1 (NTPDase1) and ecto-5'-nucleotidase were the major ectonucleotidases expressed by SMCs in the bladder. RT-qPCR revealed that, among the nucleotide receptors, there was higher expression of P2X1, P2Y1, and P2Y6 receptors. Ex vivo, nucleotides induced a more potent contraction of bladder strips isolated from NTPDase1 deficient (Entpd1-/-) mice compared to wild type controls. The strongest responses were obtained with uridine 5'-triphosphate (UTP) and uridine 5'-diphosphate (UDP), suggesting the involvement of P2Y6 receptors, which was confirmed with P2ry6-/- bladder strips. Interestingly, this response was reduced in female bladders. Our results also suggest the participation of P2X1, P2Y2 and/or P2Y4, and P2Y12 in these contractions. A reduced response to the thromboxane analogue U46619 was also observed in wild type, Entpd1-/-, and P2ry6-/- female bladders showing another difference due to sex. In summary, NTPDase1 modulates the activation of nucleotide receptors in mouse bladder SMCs, and contractions induced by P2Y6 receptor activation were weaker in female bladders.

A Simple and Efficient Genetic Immunization Protocol for the Production of Highly Specific Polyclonal and Monoclonal Antibodies against the Native Form of Mammalian Proteins.

We have generated polyclonal and monoclonal antibodies by genetic immunization over the last two decades. In this paper, we present our most successful methodology acquired over these years and present the animals in which we obtained the highest rates of success. The technique presented is convenient, easy, affordable, and generates antibodies against mammalian proteins in their native form. This protocol requires neither expensive equipment, such as a gene gun, nor sophisticated techniques such as the conjugation of gold microspheres, electroporation, or surgery to inject in lymph nodes. The protocol presented uses simply the purified plasmid expressing the protein of interest under a strong promoter, which is injected at intramuscular and intradermal sites. This technique was tested in five species. Guinea pigs were the animals of choice for the production of polyclonal antibodies. Monoclonal antibodies could be generated in mice by giving, as a last injection, a suspension of transfected cells. The antibodies detected their antigens in their native forms. They were highly specific with very low non-specific background levels, as assessed by immune-blots, immunocytochemistry, immunohistochemistry and flow cytometry. We present herein a detailed and simple procedure to successfully raise specific antibodies against native proteins.

Investigation of ammonium ion removal from aqueous solutions using arene- and propylsulfonic Acid functionalized mesoporous silica adsorbents.

To counter environmental threats to the water resources polluted by NH, which is common in wastewaters and agricultural runoff, adsorption using mesoporous functional materials represents a promising alternative to existing treatment methods. In this study, adsorption of NH ions from aqueous solutions was investigated on arene- and propylsulfonic acid functionalized SBA-15 mesoporous silica materials. The adsorbents were synthesized via co-condensation and post-synthesis grafting procedures. Adsorbents were characterized by means of X-ray diffraction, N physisorption, titration, and elemental analyses. The effects of pH, NH initial concentration, temperature, adsorbent loading, organosilane molar ratio, and presence of competitive species on the performance of the adsorbent materials were examined. All the adsorbents having an organosilane/silica molar ratio of 1:5 displayed maximum adsorption capacity around approximately 25 mg g NH at the lowest temperature investigated, 5°C. This capacity decreased with increasing temperature. For a given initial NH concentration, the removal efficiency () increased with increasing adsorbent loading. For instance, increased from 24 to 59% when the adsorbent loading was increased from 2 to 10 g L at 25°C. The adsorption isotherms were well described by a Langmuir model equation. Adsorption capacity improved with increasing organosilane/silica molar ratio, reaching 42 mg g NH with a ratio of 2:5 at 25°C. Arene- and propylsulfonic acid functionalized SBA-15 materials synthesized via co-condensation and post-synthesis grafting proved to be effective high-capacity adsorbents for the removal of NH ions from aqueous solutions.