Many membrane proteins incur a folding problem during biosynthesis; only a fraction thereof is exported from the endoplasmic reticulum (ER), because quality control is stringent. This is also true for G protein-coupled receptors. Here, we identify the deubiquitinating enzyme Usp4 as an interaction partner of the A 2a adenosine receptor, a G s -coupled receptor. Usp4 binds to the carboxyl terminus of the A 2A receptor and allows for its accumulation as deubiquinated protein. This relaxes ER quality control and enhances cell surface expression of functionally active receptor. The effect of Usp4 on the A 2A receptor was specific because 1) it was not seen in C-terminally truncated versions of the receptor; 2) it was not mimicked by Usp14, another member of the ubiquitin-specific protease family; and 3) it was not seen with the metabotropic glutamate receptor-5, another G protein-coupled receptor with a high propensity for intracellular retention. These observations show that deubiquinating enzymes can regulate quality control in the ER.
SUMMARYAquaporins (AQPs) are integral membrane channels responsible for the transport of water across a cell membrane. Based on reports that AQPs are present and accumulate in the female reproductive tract late in pregnancy, our aim was to study the expression of AQP isoforms (AQP1, 2, 3, 5, 8, and 9) at the end of pregnancy in rat in order to determine if they play a role in parturition. Reverse-transcriptase PCR revealed that specific Aqp mRNAs were detectable in the myometrium of nonpregnant and late-pregnancy (Days 18, 20, 21, and 22 of pregnancy) rat uteri. The expression of Aqp5 mRNA and protein were most pronounced on Days 18À21, and were dramatically decreased on Day 22 of pregnancy. In contrast, a significant increase was found in the level of Aqp5 transcript in whole-blood samples on the last day of pregnancy. The effect of oxytocin on myometrial Aqp5 expression in an organ bath was also investigated. The level of Aqp5 mRNA significantly decreased 5 min after oxytocin (10 À8 M) administration, similarly to its profile on the day of delivery; this effect was sensitive to the oxytocin antagonist atosiban. The vasopressin analog desmopressin (3.7 Â 10 À8 M), on the other hand, did not alter the expression of Aqp5, but did increased the amount of Aqp2 mRNA, an effect that was atosiban-resistant. These results lead us to propose that oxytocin selectively influences the expression of Aqp5 at the end of pregnancy, and may participate in events that lead to parturition in the rat. The sudden increase of AQP5 in the blood on the last day of pregnancy may serve as a marker that indicates the initiation of delivery.Mol. Reprod. Dev. 81: 524À530,
The unique requirements of poorly water-soluble drug delivery have driven a great deal of research into new formulations and routes of administration. This study investigates the use of nanosuspensions for solubility enhancement and drug delivery. Simple methods were used to prepare nasal formulations of loratadine based on nanosuspension pre-dispersion with sodium hyaluronate as a mucoadhesive agent. The nanosuspension was prepared by antisolvent precipitation method followed by ultrasonication and characterized for particle size, polydispersity index, zeta potential, morphology, and structure. Moreover, the nasal formulations were characterized for drug loading, pH, particle size, viscosity, bioadhesive viscosity parameter, and were evaluated for in vitro dissolution and diffusion, in addition to in vivo studies in a rat model. Loratadine nanosuspension displayed a particle size of 311 nm, a polydispersity index of 0.16, and zeta potential of -22.05 mV. The nanosuspension preserved the crystalline status of the raw drug. The addition of sodium hyaluronate exhibited an increase in the mean particle size and zeta potential of the nanoparticles. The nasal formulations showed enhanced bioadhesive properties compared to the unprocessed loratadine in the reference samples. The nanosuspension based-formulation that contained 5 mg mL −1 sodium hyaluronate and 2.5 mg mL −1 loratadine (NF4) showed a significant enhancement of flux and permeability coefficient through a synthetic membrane. NF4 exhibited 24.73 µg cm −2 h −1 and 0.082 cm h −1 , while the reference sample showed 1.49 µg cm −2 h −1 and 0.017 cm h −1 , for the flux and the permeability coefficient, respectively. Nasal administration of NF4 showed a bioavailability of 5.54-fold relative to the oral administration. The results obtained in this study indicate the potential of the nasal route and the nanosuspension for loratadine delivery. The relative bioavailability of NF4 was 1.84-fold compared to unprocessed loratadine in the reference sample. Therefore, the nanosized loratadine could be suggested as a practical and simple nanosystem for the intranasal delivery with improved bioavailability.
Obesity is a global health problem even among pregnant women. Obesity alters quality of labor, such as preterm labor, prolonged labor, and higher oxytocin requirements in pregnant women. The most important factors to play a role in the altered gestational period and serve as drug targets to treat the consequences are female sexual hormones, calcium channels, adrenergic system, oxytocin, and prostaglandins. However, we have limited information about the impact of obesity on the pregnant uterine contractility and gestation time. Adipose tissue, which is the largest endocrine and paracrine organ, especially in obesity, is responsible for the production of adipokines and various cytokines and chemokines, and there are no reliable data available describing the relation between body mass index, glucose intolerance, and adipokines during pregnancy. Recent data suggest that the dysregulation of leptin, adiponectin, and kisspeptin during pregnancy contributes to gestational diabetes mellitus and pre-eclampsia. A preclinical method for obese pregnancy should be developed to clarify the action of adipokines and assess their impact in obesity. The deeper understanding of the adipokines-induced processes in obese pregnancy may be a step closer to the prevention and therapy of preterm delivery or prolonged pregnancy. Gestational weight gain is one of the factors that could influence the prenatal development, birth weight, and adiposity of newborn.
Water is the major component of cells and tissues, and the movement of water across the cell membrane is a fundamental property of life. Until the discovery of the first water channel, aquaporin, it was long assumed that the transport of water was due to simple diffusion through the lipid bilayer membrane that encloses cells. Aquaporin (AQP) molecules were first discovered in the human uterus in 1994, and since then several studies have investigated these channels in the female reproductive system. The expressions of AQPs have been proven in the reproductive system. Their levels are altered during the implantation process, both in the uterus and the fetal cells, and participate in the control of the flow of amniotic fluid. They seem to be very important for the normal placental functions. AQPs are present during parturition, participating in the control of pregnant myometrial contractions and cervical ripening. However, most of the physiological and regulatory roles of AQPs are not clarified in the reproductive tract. Furthermore, no satisfactory knowledge is available about their sensitivities to different drugs. AQP-selective ligands may contribute to the development of new drug candidates and the therapy of several reproductive disorders.
A combination of selective PDE4 inhibitors and β(2) -agonists should be considered for the treatment of preterm contractions.
AimsAquaporins (AQPs) are channel proteins that facilitate the rapid passive movement of water. In our studies it was proved that the decreased AQP5 expression is followed by the increase of uterine contractility. The transient receptor potential vanilloid 4 (TRPV4) is a calcium channel, which is activated in response to osmotic changes. Our aim was to determine the possible role of AQP5 in this osmotic regulation of TRPV4, thus in pregnant uterine contraction.Main methodsWe used RT-PCR and Western blot techniques for the detection of the TRPV4 expression during pregnancy in rat uterus. The localization of AQP5 and TRPV4 was determined by immunohistochemical studies. The role of TRPV4 in uterus contraction was investigated in an isolated organ bath system. In vitro uterus contractions were stimulated with KCl and its effect was investigated with the selective TRPV4 agonist (RN1747) and antagonist (RN1734).Key findingsThe TRPV4 expression continuously increased from day 18 to the last day of pregnancy. The co-expression of TRPV4 and AQP5 in the myometrium and endometrium was determined in the late pregnant uterus. The TRPV4 antagonist and agonist significantly decreased and increased uterine contraction, respectively, especially on the last day of pregnancy.SignificanceWe presume the decreased AQP5 expression triggers hypertonic stress, which activates TRPV4 and increases uterus contraction on the day of labor. Based on these findings, we suppose the TRPV4 effect on uterus contraction is AQP5 control, which could be a new target in preterm birth therapy.
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