Search

Matheus L Medeiros, Akila L Oliveira, Mariana G de Oliveira, Fabíola Z Mónica, Edson Antunes Department of Pharmacology, University of Campinas (UNICAMP), Campinas, Sao Paulo, BrazilCorrespondence: Edson AntunesDepartment of Pharmacology, Faculty of Medical Sciences, University of Campinas (UNICAMP), Campinas, Sao Paulo, 13084-971, BrazilTel +55 19-9-9601-1516Email eantunes@unicamp.br; edson.antunes@uol.com.brPurpose: Methylglyoxal (MGO) is a highly reactive dicarbonyl species implicated in diabetic-associated diseases. Acute lung injury (ALI) symptoms and prognosis are worsened by diabetes and obesity. Here, we hypothesized that elevated MGO levels aggravate ALI, which can be prevented by metformin. Therefore, this study evaluated the lung inflammation in lipopolysaccharide (LPS)-exposed mice pretreated with MGO.Methods: C57Bl/6 male mice treated or not with MGO for 12 weeks were intranasally instilled with LPS (30 μg) to induce ALI, and metformin (300 mg/kg) was given as gavage in the last two weeks of treatment. After 6 h, bronchoalveolar lavage fluid (BALF) and lung tissues were collected to quantify the cell infiltration, cytokine levels, reactive-oxygen species (ROS) production, and RAGE expression.Results: LPS exposure markedly increased the neutrophil infiltration in BALF and lung tissue, which was accompanied by higher levels of IFN-γ, TNF-α and IL-1β compared with untreated group. MGO treatment significantly increased the airways neutrophil infiltration and mRNA expressions of TNF-α and IL-1β, whereas COX-2 expression remained unchanged. In lung tissues of LPS-exposed mice, MGO treatment significantly increased the immunostaining and mRNA expression of RAGE, and the ROS levels. Serum MGO concentration achieved after 12-week intake was 9.2-fold higher than control mice, which was normalized by metformin treatment. Metformin also reduced the inflammatory markers in response to MGO.Conclusion: MGO intake potentiates the LPS-induced ALI, increases RAGE expression and ROS generation, which is normalized by metformin. MGO scavengers may be a good adjuvant therapy to reduce ALI in patients with cardiometabolic diseases.Keywords: advanced glycated end products, neutrophil, airways, immunohistochemistry

Multidrug resistance proteins type 4 (MRP4) and 5 (MRP5) play pivotal roles in the transport of cyclic nucleotides in various tissues. However, their specific functions within the lower urinary tract remain relatively unexplored. This study aimed to investigate the effect of pharmacological inhibition of MRPs on cyclic nucleotide signaling in isolated pig bladder. The relaxation responses of the bladder were assessed in the presence of the MRP inhibitor, MK571. The temporal changes in intra- and extracellular levels of cAMP and cGMP in stimulated tissues were determined by mass spectrometry. The gene ( ABCC4 ) and protein (MRP4) expression were also determined. MK571 administration resulted in a modest relaxation effect of approximately 26% in carbachol-precontracted bladders. The relaxation induced by phosphodiesterase inhibitors such as cilostazol, tadalafil, and sildenafil was significantly potentiated in the presence of MK571. In contrast, no significant potentiation was observed in the relaxation induced by substances elevating cAMP levels or stimulators of soluble guanylate cyclase. Following forskolin stimulation, both intracellular and extracellular cAMP concentrations increased by approximately 15.8-fold and 12-fold, respectively. Similarly, stimulation with tadalafil + BAY 41-2272 resulted in roughly 8.2-fold and 3.4-fold increases in intracellular and extracellular cGMP concentrations, respectively. The presence of MK571 reduced only the extracellular levels of cGMP. This study reveals the presence and function of MRP4 transporters within the porcine bladder and paves the way for future research exploring the role of this transporter in both underactive and overactive bladder disorders. NEW & NOTEWORTHY This study investigates the impact of pharmacological inhibition of MRP4 and MRP5 transporters on cyclic nucleotide signaling in isolated pig bladders. MK571 administration led to modest relaxation, with enhanced effects observed in the presence of phosphodiesterase inhibitors. However, substances elevating cAMP levels remained unaffected. MK571 selectively reduced extracellular cGMP levels. These findings shed light on the role of MRP4 transporters in the porcine bladder, opening avenues for further research into bladder disorders.

Methylglyoxal (MGO) is a highly reactive α-dicarbonyl compound formed endogenously from 3-carbon glycolytic intermediates. Methylglyoxal accumulated in plasma and urine of hyperglycemic and diabetic individuals acts as a potent peptide glycation molecule, giving rise to advanced glycation end products (AGEs) like arginine-derived hydroimidazolone (MG-H1) and carboxyethyl-lysine (CEL). Methylglyoxal-derived AGEs exert their effects mostly via activation of RAGE, a cell surface receptor that initiates multiple intracellular signaling pathways, favoring a pro-oxidant environment through NADPH oxidase activation and generation of high levels of reactive oxygen species (ROS). Diabetic bladder dysfunction is a bothersome urological complication in patients with poorly controlled diabetes mellitus and may comprise overactive bladder, urge incontinence, poor emptying, dribbling, incomplete emptying of the bladder, and urinary retention. Preclinical models of type 1 and type 2 diabetes have further confirmed the relationship between diabetes and voiding dysfunction. Interestingly, healthy mice supplemented with MGO for prolonged periods exhibit in vivo and in vitro bladder dysfunction, which is accompanied by increased AGE formation and RAGE expression, as well as by ROS overproduction in bladder tissues. Drugs reported to scavenge MGO and to inactivate AGEs like metformin, polyphenols, and alagebrium (ALT-711) have shown favorable outcomes on bladder dysfunction in diabetic obese leptin-deficient and MGO-exposed mice. Therefore, MGO, AGEs, and RAGE levels may be critically involved in the pathogenesis of bladder dysfunction in diabetic individuals. However, there are no clinical trials designed to test drugs that selectively inhibit the MGO-AGEs-RAGE signaling, aiming to reduce the manifestations of diabetes-associated bladder dysfunction. This review summarizes the current literature on the role of MGO-AGEs-RAGE-ROS axis in diabetes-associated bladder dysfunction. Drugs that directly inactivate MGO and ameliorate bladder dysfunction are also reviewed here.

Introduction: The transient receptor potential ankyrin 1 channel (TRPA1) is expressed in urothelial cells and bladder nerve endings. Hyperglycemia in diabetic individuals induces accumulation of the highly reactive dicarbonyl compound methylglyoxal (MGO), which modulates TRPA1 activity. Long-term oral intake of MGO causes mouse bladder dysfunction. We hypothesized that TRPA1 takes part in the machinery that leads to MGO-induced bladder dysfunction. Therefore, we evaluated TRPA1 expression in the bladder and the effects of 1 h-intravesical infusion of the selective TRPA1 blocker HC-030031 (1 nmol/min) on MGO-induced cystometric alterations. Methods: Five-week-old female C57BL/6 mice received 0.5% MGO in their drinking water for 12 weeks, whereas control mice received tap water alone. Results: Compared to the control group, the protein levels and immunostaining for the MGO-derived hydroimidazolone isomer MG-H1 was increased in bladders of the MGO group, as observed in urothelium and detrusor smooth muscle. TRPA1 protein expression was significantly higher in bladder tissues of MGO compared to control group with TRPA1 immunostaining both lamina propria and urothelium, but not the detrusor smooth muscle. Void spot assays in conscious mice revealed an overactive bladder phenotype in MGO-treated mice characterized by increased number of voids and reduced volume per void. Filling cystometry in anaesthetized animals revealed an increased voiding frequency, reduced bladder capacity, and reduced voided volume in MGO compared to vehicle group, which were all reversed by HC-030031 infusion. Conclusion: TRPA1 activation is implicated in MGO-induced mouse overactive bladder. TRPA1 blockers may be useful to treat diabetic bladder dysfunction in individuals with high MGO levels., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Oliveira, Medeiros, Gomes, Mello, Costa, Mónica and Antunes.)

Glycolytic overload in diabetes causes large accumulation of the highly reactive dicarbonyl compound methylglyoxal (MGO) and overproduction of advanced glycation end products (AGEs), which interact with their receptors (RAGE), leading to diabetes-associated macrovascular complications. The bladder is an organ that stays most in contact with dicarbonyl species, but little is known about the importance of the MGO-AGEs-RAGE pathway to diabetes-associated bladder dysfunction. Here, we aimed to investigate the role of the MGO-AGEs-RAGE pathway in bladder dysfunction of diabetic male and female ob / ob mice compared with wild-type (WT) lean mice. Diabetic ob / ob mice were treated with the AGE breaker alagebrium (ALT-711, 1 mg/kg) for 8 wk in drinking water. Compared with WT animals, male and female ob / ob mice showed marked hyperglycemia and insulin resistance, whereas fluid intake remained unaltered. Levels of total AGEs, MGO-derived hydroimidazolone 1, and RAGE in bladder tissues, as well as fluorescent AGEs in serum, were significantly elevated in ob / ob mice of either sex. Collagen content was also markedly elevated in the bladders of ob / ob mice. Void spot assays in filter paper in conscious mice revealed significant increases in total void volume and volume per void in ob / ob mice with no alterations of spot number. Treatment with ALT-711 significantly reduced the levels of MGO, AGEs, RAGE, and collagen content in ob / ob mice. In addition, ALT-711 treatment normalized the volume per void and increased the number of spots in ob / ob mice. Activation of AGEs-RAGE pathways by MGO in the bladder wall may contribute to the pathogenesis of diabetes-associated bladder dysfunction. NEW & NOTEWORTHY The involvement of methylglyoxal (MGO) and advanced glycation end products (AGEs) in bladder dysfunction of diabetic ob/ob mice treated with the AGE breaker ALT-711 was investigated here. Diabetic mice exhibited high levels of MGO, AGEs, receptor for AGEs (RAGE), and collagen in serum and/or bladder tissues along with increased volume per void, all of which were reduced by ALT-711. Activation of the MGO-AGEs-RAGE pathway in the bladder wall contributes to the pathogenesis of diabetes-associated bladder dysfunction.

Aims: Melatonin is known to inhibit platelet aggregation induced by arachidonic acid (AA). In the present study we investigated whether agomelatine (Ago), an antidepressant with agonist activity at melatonin receptor 1 (MT1) and MT2 could reduce platelets aggregation and adhesion., Main Methods: Human platelets from healthy donors were used to test the in vitro effects of Ago in the presence of different platelet activators. We performed aggregation and adhesion assays, thromboxane B 2 (TxB 2 ), cAMP and cGMP measurements, intra-platelet calcium registration and flow cytometry assays., Key Findings: Our data revealed that different concentrations of Ago reduced AA- and collagen-induced human platelet aggregation in vitro. Ago also reduced AA-induced increase in thromboxane B 2 (TxB 2 ) production, intracellular calcium levels and P-selectin expression at plasma membrane. The effects of Ago in AA-activated platelets were likely dependent on MT1 as they were blocked by luzindole (a MT1/MT2 antagonist) and mimicked by the MT1 agonist UCM871 in a luzindole-sensitive manner. The MT2 agonist UCM924 was also able to inhibit platelet aggregation, but this response was not affected by luzindole. On the other hand, although UCM871 and UCM924 reduced collagen-induced platelet aggregation and adhesion, inhibition of collagen-induced platelet aggregation by Ago was not mediated by melatonin receptors because it was not affected by luzindole., Significance: The present data show that Ago suppresses human platelet aggregation and suggest that this antidepressant may have the potential to prevent atherothrombotic ischemic events by reducing thrombus formation and vessel occlusion., Competing Interests: Declaration of competing interest Gabriella Gobbi is an inventor of patents on selective melatonin MT2 ligands., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: The prostate gland is surrounded by periprostatic adipose tissue (PPAT) that can release mediators that interfere in prostate function. In this study, we examined the effect of periprostatic adipose tissue supernatant obtained from obese mice on prostate reactivity in vitro and on the viability of human prostatic epithelial cell lines. Methods: Male C57BL/6 mice were fed a standard or high-fat diet after which PPAT was isolated, incubated in Krebs-Henseleit solution for 30 min (without prostate) or 60 min (with prostate), and the supernatant was then collected and screened for biological activity. Total nitrate and nitrite (NOx - ) and adenosine were quantified, and the supernatant was then collected and screened for biological activity. NOx - and adenosine were quantified. Concentration-response curves to phenylephrine (PE) were obtained in prostatic tissue from lean and obese mice incubated with or without periprostatic adipose tissue. In some experiments, periprostatic adipose tissue was co-incubated with inhibitors of the nitric oxide (NO)-cyclic guanosine monophosphate pathway (L-NAME, 1400W, ODQ), adenylate cyclase (SQ22536) or with adenosine A 2A (ZM241385), and A 2B (MRS1754) receptor antagonists. PNT1-A (normal) and BPH-1 (hyperplasic) human epithelial cells were cultured and incubated with supernatant from periprostatic adipose tissue for 24, 48, or 72 h in the absence or presence of these inhibitors/antagonists, after which cell viability and proliferation were assessed. Results: The levels of NOx - and adenosine were significantly higher in the periprostatic adipose tissue supernatant (30 min, without prostate) when compared to the vehicle. A trend toward an increase in the levels of NOX was observed after 60 min. PPAT supernatant from obese mice significantly reduced the PE-induced contractions only in prostate from obese mice. The co-incubation of periprostatic adipose tissue with L-NAME, 1400W, ODQ, or ZM241385 attenuated the anticontractile activity of the periprostatic adipose tissue supernatant. Incubation with the supernatant of periprostatic adipose tissue from obese mice significantly increased the viability of PNT1-A cells and attenuated expression of the apoptosis marker protein caspase-3 when compared to cells incubated with periprostatic adipose tissue from lean mice. Hyperplastic cells (BPH-1) incubated with periprostatic adipose tissue from obese mice showed greater proliferation after 24 h, 48 h, and 72 h compared to cells incubated with culture medium alone. BPH-1 cell proliferation in the presence of PPAT supernatant was attenuated by NO-signaling pathway inhibitors and by adenosine receptor antagonists after 72 h. Conclusion: NO and adenosine are involved in the anticontractile and pro-proliferative activities of periprostatic adipose tissue supernatant from obese mice. More studies are needed to determine whether the blockade of NO and/or adenosine derived from periprostatic adipose tissue can improve prostate function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Passos, de Oliveira, Ghezzi, Mello, Levi D’Ancona, Teixeira, Muscará, Grespan Bottoli, Vilela de Melo, de Oliveira, Antunes and Mónica.)

6-Nitrodopamine is a novel catecholamine released by vascular tissues, heart, and vas deferens. The aim of this study was to investigate whether 6-nitrodopamine is released from the thoracic aorta and pulmonary artery rings of marmosets (Callithrix spp.) and to evaluate the relaxing and anti-contractile actions of this catecholamine. Release of 6-nitrodopamine, dopamine, noradrenaline, and adrenaline was assessed by liquid chromatography with tandem mass spectrometry (LC-MS/MS). The relaxations induced by 6-nitrodopamine and by the selective dopamine D2 receptor antagonist L-741,626 were evaluated on U-46619 (3 nM)-pre-contracted vessels. The effects of 6-nitrodopamine and L-741,626 on the contractions induced by electric-field stimulation (EFS), dopamine, noradrenaline, and adrenaline were also investigated. Both aorta and pulmonary artery rings exhibited endothelium-dependent release of 6-nitrodopamine, which was significantly reduced by the NO synthesis inhibitor L-NAME. Addition of 6-nitrodopamine or L-741,626 caused concentration-dependent relaxations of both vascular tissues, which were almost abolished by endothelium removal, whereas L-NAME and the soluble guanylate cyclase inhibitor ODQ had no effect on 6-nitrodopamine-induced relaxations. Additionally, pre-incubation with 6-nitrodopamine antagonized the dopamine-induced contractions, without affecting the noradrenaline- and adrenaline-induced contractions. Pre-incubation with L-741,626 antagonized the contractions induced by all catecholamines. The EFS-induced contractions were significantly increased by L-NAME, but unaffected by ODQ. Immunohistochemical assays showed no immunostaining of the neural tissue markers S-100 and calretinin in either vascular tissue. The results indicated that 6-nitrodopamine is the major catecholamine released by marmoset vascular tissues, and it acts as a potent and selective antagonist of dopamine D2-like receptors. 6-nitrodopamine release may be the major mechanism by which NO causes vasodilatation.

Malignant glioma is the most common and deadly brain tumor. A marked reduction in the levels of sGC (soluble guanylyl cyclase) transcript in the human glioma specimens has been revealed in our previous studies. In the present study, restoring the expression of sGCβ1 alone repressed the aggressive course of glioma. The antitumor effect of sGCβ1 was not associated with enzymatic activity of sGC since overexpression of sGCβ1 alone did not influence the level of cyclic GMP. Additionally, sGCβ1-induced inhibition of the growth of glioma cells was not influenced by treatment with sGC stimulators or inhibitors. The present study is the first to reveal that sGCβ1 migrated into the nucleus and interacted with the promoter of the TP53 gene. Transcriptional responses induced by sGCβ1 caused the G0 cell cycle arrest of glioblastoma cells and inhibition of tumor aggressiveness. sGCβ1 overexpression impacted signaling in glioblastoma multiforme, including the promotion of nuclear accumulation of p53, a marked reduction in CDK6, and a significant decrease in integrin α6. These anticancer targets of sGCβ1 may represent clinically important regulatory pathways that contribute to the development of a therapeutic strategy for cancer treatment.

Background: Intracellular levels of cyclic nucleotides can also be controlled by the action of multidrug resistance protein types 4 (MRP4) and 5 (MRP5). To date, no studies evaluated the role of their inhibition in an animal model of erectile dysfunction (ED)., Objectives: To evaluate the effect of a 2-week treatment with MK571, an inhibitor of the efflux of cyclic nucleotides in the ED of obese mice., Materials and Methods: Mice were divided in three groups: (i) lean, (ii) obese, and (iii) obese + MK571. The corpus cavernosum (CC) were isolated, and concentration-response curves to acetylcholine (ACh), sodium nitroprusside (SNP), and tadalafil in addition to electrical field stimulation (EFS) were carried out in phenylephrine pre-contracted tissues. Expression of ABCC4 and ABCC5, intracellular levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), the protein levels for pVASP Ser157 and pVASP Ser239 , and the intracavernous pressure (ICP) were also determined. The intracellular and extracellular (supernatant) ratios in CC from obese and lean stimulated with a cGMP-increasing substance (BAY 58-2667) in the absence and presence of MK571 (20 μM, 30 min) were also assessed., Results: The treatment with MK571 completely reversed the lower relaxing responses induced by EFS, ACh, SNP, and tadalafil observed in obese mice CC in comparison with untreated obese mice. Cyclic GMP and p-VASP Ser239 expression were significantly reduced in CC from obese groups. MK571 promoted a sixfold increase in cGMP without interfering in the protein expression of p-VASP Ser239 . Neither the cAMP levels nor p-VASP Ser157 were altered in MK571-treated animals. The ICP was ∼50% lower in obese than in the lean mice; however, the treatment with MK571 fully reversed this response. Expressions of ABCC4 and ABCC5 were not different between groups. The intra/extracellular ratio of cGMP was similar in CC from obese and lean mice stimulated with BAY 58-2667., Conclusions: The MRPs inhibition by MK571 favored the accumulation of cGMP in the smooth muscle cells, thus improving the smooth muscle relaxation and the erectile function in obese mice., (© 2022 American Society of Andrology and European Academy of Andrology.)

The development of essential hypertension involves several factors. Vascular dysfunction, characterized by endothelial dysfunction, low-grade inflammation and structural remodeling, plays an important role in the initiation and maintenance of essential hypertension. Although the mechanistic pathways by which essential hypertension develops are poorly understood, several pharmacological classes available on the clinical settings improve blood pressure by interfering in the cardiac output and/or vascular function. This review is divided in two major sections. The first section depicts the major molecular pathways as renin angiotensin aldosterone system (RAAS), endothelin, nitric oxide signalling pathway and oxidative stress in the development of vascular dysfunction. The second section describes the role of some pharmacological classes such as i) RAAS inhibitors, ii) dual angiotensin receptor-neprilysin inhibitors, iii) endothelin-1 receptor antagonists, iv) soluble guanylate cyclase modulators, v) phosphodiesterase type 5 inhibitors and vi) sodium-glucose cotransporter 2 inhibitors in the context of hypertension. Some classes are already approved in the treatment of hypertension, but others are not yet approved. However, due to their potential benefits these classes were included., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

6-Nitrodopamine (6-ND) is released by rat vas deferens and exerts a potent contractile response that is antagonized by tricyclic antidepressants and α 1 -, β 1 - and β 1 /β 2 -adrenoceptor antagonists. The release of 6-ND, noradrenaline, adrenaline and dopamine from rat isolated right atria was assessed by tandem mass spectrometry. The effects of the catecholamines were evaluated in both rat isolated right atria and in anaesthetized rats. 6-ND was the major catecholamine released from the isolated atria and the release was significantly reduced in nitric oxide synthase inhibitor L-NAME pre-treated atria or in atria obtained from L-NAME chronically treated animals, but unaffected by tetrodotoxin. 6-ND (1 pM) significantly increased the atrial frequency, being 100 times more potent than noradrenaline and adrenaline. Selective β 1 -blockers reduced the atrial frequency only at concentrations that prevented the increases in atrial frequency induced by 6-ND 1pM. Conversely, β 1 -blockade did not affect dopamine (10 nM), noradrenaline (100 pM) or adrenaline (100 pM) effect. The reductions in atrial frequency induced by the β 1 -adrenoceptor antagonists were absent in L-NAME pre-treated atria and in atria obtained from chronic L-NAME-treated animals. Tetrodotoxin did not prevent the reduction in atrial frequency induced by L-NAME or by β 1 -blockers treated preparations. In anaesthetized rats, at 1 pmol/kg, only 6-ND caused a significant increase in heart rate. Inhibition of 6-ND synthesis by chronic L-NAME treatment reduced both atrial frequency and heart rate. The results indicate that 6-ND is a major modulator of rat heart chronotropism and the reduction in heart rate caused by β 1 -blockers are due to selective blockade of 6-ND receptor., Competing Interests: Declaration of competing interest Authors declare that they have no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

6-Nitrodopamine (6-ND) is an endogenous modulator of the contractility in the rat isolated epididymal vas deferens (RIEVD) and considered to be the main peripheral mediator of the emission process. Use of selective and unselective β-adrenergic receptor antagonists has been associated with ejaculatory failure. Here, the effects of selective β 1 - and β 1 /β 2 -adrenergic receptor antagonists on RIEVD contractions induced by 6-ND, dopamine, noradrenaline, adrenaline, and electric-field stimulation (EFS) were investigated. The selective β 1 -adrenergic receptor antagonists atenolol (0.1 and 1 µM), betaxolol (1 µM), and metoprolol (1 µM) and the unselective β 1 /β 2 -adrenergic receptor antagonists propranolol (1 and 10 µM) and pindolol (10 µM) caused significant rightward shifts of the concentration-response curve to 6-ND (pA 2 6.41, 6.91, 6.75, 6.47, and 5.74; for atenolol, betaxolol, metoprolol, propranolol, and pindolol), but had no effect on dopamine-, noradrenaline-, and adrenaline-induced contractions. The effects of selective β 1 - and β 1 /β 2 -adrenergic receptor antagonists at a higher concentration (atenolol 1 µM, betaxolol 1 µM, metoprolol 1 µM, propranolol 10 µM, and pindolol 10 µM) also reduced the EFS-induced RIEVD contractions in control, but not in RIEVD obtained from L-NAME-treated animals. The selective β 1 -adrenoceptor agonist RO-363, the selective β 2 -adrenoceptor agonist salbutamol, and the selective β 3 -adrenoceptor agonist mirabegron, up to 300 µM, had no effect on the RIEVD tone. The results demonstrate that β 1 - and β 1 -/β 2 -adrenoceptor receptor antagonists act as 6-ND receptor antagonists in RIEVD, further confirming the main role of 6-ND in the RIEVD contractility., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Chelonoidis carbonaria aortic rings present endothelium-derived release of dopamine, noradrenaline, adrenaline and 6-nitrodopamine (6-ND). Here it was investigated whether 6-ND release is coupled to nitric oxide (NO) synthesis and its action on the vascular smooth muscle reactivity. Basal release of 6-ND from aortic rings in the absence and presence of the NO synthesis inhibitor L-NAME was quantified by LC-MS-MS. Aortic rings were suspended vertically between two metal hooks in 10-mL organ baths containing Krebs-Henseleit's solution and attached to isometric transducers. The tissues were allowed to equilibrate for 1 h before starting the experiments. The release of 6-ND was significantly reduced by previous incubation with L-NAME. 6-ND (up to 300 μM) had no contractile activity in the aortic rings. 6-ND (1, 3 and 10 μM) produced significant rightward shifts of the concentration-response curves to dopamine in endothelium-intact (pA 2 6.09) and L-NAME pre-treated endothelium-intact (pA 2 7.06) aortic rings. Contractions induced by noradrenaline and adrenaline were not affected by pre-incubation with 6-ND. The EFS (16 Hz)-induced aortic contractions were significantly inhibited by incubation with 6-ND (10 μM). In the thromboxane A 2 mimetic U-46619 (30 nM) pre-contracted endothelium intact aortic rings, 6-ND (1 nM-1 μM) and the dopamine D 2 -receptor antagonist haloperidol (1 nM-1 μM) induced concentration-dependent relaxations. The relaxations were not present in endothelium-removed aortic rings but they were not affected by incubation with L-NAME in endothelium-intact aortic rings. The results indicate that the synthesis of this novel catecholamine in Chelonoidis carbonaria aortic rings is coupled to NO release and that 6-ND acts as a highly selective dopamine D 2 -like receptor antagonist., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Methylglyoxal (MGO) is a highly reactive dicarbonyl compound implicated in diabetes-associated diseases. In vascular tissues, MGO induces the formation of advanced glycation end products (AGEs) that bounds its receptor RAGE, initiating the downstream tissue injury. Outside the cardiovascular system, MGO intake produces mouse voiding dysfunction and bladder overactivity. We have sought that MGO-induced bladder overactivity is due to activation of AGE-RAGE-reactive-oxygen species (ROS) signaling cascade, leading to Rho kinase activation. Therefore, female mice received 0.5% MGO orally for 12 weeks, after which in vitro bladder contractions were evaluated in the presence or not of superoxide dismutase (PEG-SOD) or the Rho kinase inhibitor Y27632. Treatment with MGO significantly elevated the serum levels of MGO and fluorescent AGEs, as well as the RAGE immunostaining in the urothelium, detrusor, and vascular endothelium. RAGE mRNA expression in the bladder was also higher in the MGO group. Methylglyoxal significantly increased the ROS production in both urothelium and detrusor smooth muscle, with the increases in detrusor markedly higher than urothelium. The bladder activity of superoxide dismutase (SOD) was significantly reduced in the MGO group. Gene expressions of L-type Ca 2+ channels, RhoA, ROCK-1, and ROCK-2 in bladder tissues were significantly elevated in the MGO group. Increased bladder contractions to electrical-field stimulation, carbachol α,β-methylene ATP, and extracellular Ca 2+ were observed after MGO exposure, which was significantly reduced by prior incubation with either PEG-SOD or Y27632. Overall, our data indicate serum MGO accumulation elevates the AGEs levels and activates the RAGE-ROS signaling leading to Rho kinase-induced muscle sensitization, ultimately leading to detrusor overactivity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Oliveira, Medeiros, de Oliveira, Teixeira, Mónica and Antunes.)

6-nitrodopamine (6-ND) is released from rat isolated vas deferens and modulates electrical-field stimulation (EFS) contractions of the rat isolated epididymal vas deferens (RIEVD) via a specific receptor which is blocked by tricyclic antidepressants. Here, the effects of selective α 1 -adrenergic receptor antagonists on RIEVD contractions induced by 6-ND, dopamine, noradrenaline, adrenaline and EFS were investigated. Doxazosin and tamsulosin (3-10 nM) caused significant rightward shifts of the concentration-response curve to 6-ND, but had no effect on dopamine-, noradrenaline- and adrenaline-induced contractions. Alfuzosin (10 nM) produced rightward shifts on concentration-response curves to all catecholamines. Silodosin (10 nM) and terazosin (100 nM) displaced to the right the noradrenaline, dopamine and adrenaline curves, but higher concentrations of both antagonists (100 and 300 nM, respectively) were required to displace the 6-ND curves. The EFS-induced contractions were significantly inhibited only at the concentrations that the α 1 -adrenergic receptor antagonists caused rightward shifts on the 6-ND concentration-response curves. The inhibition of EFS-induced contractions by doxazosin (10 nM), tamsulosin (10 nM), alfuzosin (10 nM), silodosin (100 nM) and terazosin (300 nM), were not observed in RIEVD obtained from animals chronically treated with L-NAME. This work demonstrates that α 1 -adrenoceptor antagonists act as 6-ND receptor antagonists in RIEVD, opening the possibility that many actions previously attributed to noradrenaline could be due to 6-ND antagonism. In addition, blockade of the 6-ND receptors by both tricyclic antidepressants and α 1 -adrenergic receptor antagonists may represent the common mechanism of action responsible for their therapeutic use in the treatment of premature ejaculation., (Copyright © 2021 Elsevier B.V. All rights reserved.)