Glycine Effect on the Expression Profile of Orphan Receptors GPR21, GPR26, GPR39, GPR82 and GPR6 in a Model of Inflammation in 3T3-L1 Cells.

BACKGROUND: Chronic or low-grade inflammation is a process where various immune cells are recruited from the periphery into adipose tissue. This event gives rise to localised inflammation, in addition to having a close interaction with cardiometabolic pathologies where the mediation of orphan receptors is observed. The aim of this study was to analyse the participation of the orphan receptors GPR21, GPR39, GPR82 and GPR6 in a chronic inflammatory process in 3T3-L1 cells. The 3T3-L1 cells were stimulated with TNF-α (5 ng/mL) for 60 min as an inflammatory model. Gene expression was measured by RT-qPCR. RESULTS: We showed that the inflammatory stimulus of TNF-α in adipocytes decreased the expression of the orphan receptors GPR21, GPR26, GPR39, GPR82 and GPR6, which are related to low-grade inflammation. CONCLUSIONS: Our results suggest that GPR21 and GPR82 are modulated by glycine, it shows a possible protective role in the presence of an inflammatory environment in adipocytes, and they could be a therapeutic target to decrease the inflammation in some diseases related to low-grade inflammation such as diabetes, obesity and metabolic syndrome.

siRNA knockdown of angiopoietin 2 significantly reduces neovascularization in diabetic rats.

Diabetes is a disease that leads to proliferative diabetic retinopathy (PDR), which is associated with an increase of new vessels formation due to an overexpression of angiogenic factors, such as angiopoietin 2 (ANGPT2). The aim of this work was to design a siRNA targeting ANGPT2 to decrease the retinal neovascularization associated with PDR. Adult male Wistar rats weighing 325-375 g were used. Diabetes was induced by a single dose of streptozotocin (STZ, 60 mg/kg i.p.). The siRNAs were designed, synthesised, and administered intravitreally at the beginning of diabetes induction (t0), and after 4 weeks of diabetes evolution (t4), subsequently evaluated the retinal neovascularization (junctions and lacunarity) and ANGPT2 expression in the retina by RT-PCR, after 4 weeks of the siRNAs administration. The results showed that the administration of STZ produced significant increases in blood glucose levels, retinal neovascularization (augmented junctions and lower lacunarity), and ANGPT2 expression, while the administration of the ANGPT2-siRNAs at different groups (t0 and t4) reduces the junctions and increases the lacunarity in diabetic rats. Therefore, we conclude that the administration of siRNAs targeting ANGPT2 could be an option to decrease the retinal neovascularization associated with PDR and halt the progression of blindness caused by diabetes.

Autonomic dysregulation at multiple sites is implicated in age-associated underactive bladder in female mice.

AIMS: To evaluate the functional and molecular alterations of contractile and relaxant machinery in the bladder and urethra that lead to the underactive bladder (UAB) in old female mice. METHODS: Female young (3-months) and old (18-months) C57BL/6 mice were used. Urodynamic was assessed in awake and anaesthetized mice. Electrical-field stimulation (EFS) and concentration-response curves to contractile and relaxing agents in isolated bladders and urethras were performed. Messenger RNA (mRNA) expressions of muscarinic, adrenergic, and transient receptor potential vanilloid-4 (TRPV4), and of the enzymes tyrosine hydroxylase and neuronal nitric oxide synthase (nNOS) were determined. Bladder cyclic adenosine monophosphate (cAMP) levels were measured. RESULTS: Cystometry in old mice showed incapacity to produce bladder emptying. On filter paper, old mice showed reduced urinary spots. Compared to the young group, bladder contractions induced by EFS and carbachol were lower in old mice. The ß3 -adrenoceptor agonist mirabegron promoted higher bladder relaxation and elevation of cAMP levels in old mice. In old mice urethras, the α1a -adrenoceptor agonist phenylephrine produced higher contractions, but no differences were found for the NO donor sodium nitroprusside-induced relaxations. In old mice, increased mRNA expressions of ß3 - and α1a -adrenoceptors in bladder and urethra were found, respectively, whereas the muscarinic M2 and M3 receptors and ß2 -adrenoceptors did not change between groups. Reduced mRNA expressions of tyrosine hydroxylase and nNOS were found in old mouse urethras. Additionally, TRPV4 expression was reduced in bladder urothelium from old mice. CONCLUSION: Age-associated mouse UAB is the result of autonomic dysfunction at multiple levels leading to the less sensitive and overrelaxed bladder, along with urethral hypercontractility.

Menthol ameliorates voiding dysfunction in types I and II diabetic mouse model.

AIMS: Overactive bladder (OAB) is one of the most common complications of both type 1 (T1DM) and type 2 diabetes mellitus (T2DM). In healthy conditions, menthol infused intravesically reduces the threshold for initiating micturition reflex, but no study evaluated its effects in diabetic conditions. Therefore, we have used mouse models of T1DM and T2DM to evaluate the effects of menthol on cystometric alterations and increased bladder contractility in vitro. METHODS: For T1DM induction, male C57BL6 mice were injected with streptozotocin (STZ) and evaluated after 4 weeks. For T2DM induction, mice were fed with high-fat diet (HFD) for 12 weeks to induce obesity. Urodynamic profiles were assessed by filling cystometry through the infusion of menthol (100 µM for 30 min) or vehicle (DMSO 0.1%). Contractile responses to carbachol, potassium chloride (KCl), and electrical-field stimulation (EFS) were measured in isolated bladders after 20 min incubation with menthol (100 µM) or vehicle. RESULTS: Filling cystometry showed that STZ-injected mice exhibited higher bladder capacity, threshold pressure, and non-voiding contractions (NVCs), which were significantly reduced by menthol infusion. The increased voiding frequency in STZ group were unaffected by menthol. In HFD-fed obese mice menthol significantly attenuated the increased threshold pressure and NVC frequency, but unaffected the changes of voiding frequency. In both STZ-injected and HFD-fed mice, incubation of isolated bladders with menthol normalized the enhanced contractile responses to carbachol, KCl, and EFS stimulation. CONCLUSIONS: Menthol may be a potential pharmacological option for the treatment of OAB as a consequence of T1DM and T2DM.

Micturition dysfunction in four-month old ovariectomized rats: Effects of testosterone replacement.

AIMS: Androgen deficiency has been implicated in urological complications of postmenopausal women. This study examined the effects of testosterone replacements on the lower urinary tract dysfunction in 4-month old ovariectomized (OVX) rats. MAIN METHODS: Sprague-Dawley female rats were OVX bilaterally. Three months later, rats received single intramuscular injections of testosterone undecanoate. Cystometric study, and bladder and urethra smooth muscle reactivities were evaluated. KEY FINDINGS: Ovariectomy reduced by 65% (p<0.05) the serum testosterone levels. Testosterone replacement at 5mg/kg restored serum hormone levels to baseline, whereas 10mg/kg produced 14-fold higher testosterone levels. OVX rats exhibited significant increases of body weight, perigonadal fat and blood pressure, and reduced uterus weight, but none of these parameters were changed by testosterone replacements. OVX rats exhibited micturition dysfunction characterized by increases of basal pressure, threshold pressure, voiding frequency and post-voiding pressure. In addition, the bladder contractions induced by electrical-field stimulation (EFS) and carbachol were significantly reduced, whereas angiotensin II-induced urethral contractions were significantly increased in OVX rats. Testosterone replacement at 10mg/kg (but not at 5mg/kg) dose fully normalized the in vivo micturition dysfunction, as well as the in vitro bladder and urethral alterations. Testosterone (10mg/kg) also significantly potentiated the bladder relaxations induced by the ß3-adrenoceptor agonist mirabegron. The protective effects of testosterone were not modified by concomitant treatment with the aromatase inhibitor letrozole (2.5mg/kg, 4weeks). SIGNIFICANCE: The improvement of micturition dysfunction by testosterone replacement suggests that androgen therapy might be of therapeutic benefit for urological complications associated with post-menopause.