Effects of Local Vasodilators and the Autonomic Nervous System on Microcirculation and Mitochondrial Function in Septic Rats.

Systemic vasodilating agents like nitroglycerin (NG) or iloprost (Ilo) show beneficial effects on intestinal microcirculation during sepsis, which could be attenuated by activation of the sympathetic nervous system or systemic side effects of vasodilating agents. This exploratory study aimed to investigate the effects of topically administered vasodilators and the parasympathetic drug carbachol on colonic microcirculatory oxygenation (µHbO2), blood flow (µFlow) and mitochondrial respiration. A total of 120 male Wistar rats were randomly assigned to twelve groups and underwent either colon ascendens stent peritonitis (CASP) or sham surgery. After 24 h, animals received the following therapeutic regimes: (1) balanced full electrolyte solution, (2) carbachol, (3) NG, (4) Ilo, (5) NG + carbachol, and (6) Ilo + carbachol. Mitochondrial respiration was measured in colon homogenates by respirometry. In sham animals, NG (-13.1%*) and Ilo (-10.5%*) led to a decrease in µHbO2. Additional application of carbachol abolished this effect (NG + carbachol: -4.0%, non-significant; Ilo + carbachol: -1.4%, non-significant). In sepsis, carbachol reduced µHbO2 when applied alone (-10.5%*) or in combination with NG (-17.6%*). Thus, the direction and degree of this effect depend on the initial pathophysiologic condition.

(-)-Carvone Inhibits Oxytocin-induced Writhing Via Uterine Relaxation in Rodents.

(-)-Carvone, a ketone monoterpene, is the main component of essential oils from several medicinal plants and has been reported to have anti-arthriric, anticonvulsive, antidiabetic, anti-inflammatory, anticancer, and immunomodulatory effects. Therefore, this study aimed to investigate the spasmolytic activity of (-)-carvone in rodent models. The isolated virgin rat uterus was mounted in an organ bath apparatus, and the relaxing effect of ( -)-carvone and its mechanism of action were evaluated in tonic contractions induced by carbachol, KCl, PGF2α, or oxytocin. The animal model of primary dysmenorrhea was replicated with the injection of estradiol benzoate in female mice for three consecutive days, followed by intraperitoneal administration of oxytocin. Non-clinical acute toxicity evaluation was also performed. (-)-Carvone potency and effectiveness were larger in carbachol (pEC50 = 5.41 ± 0.14 and Emax = 92.63 ± 1.90% at 10-3 M) or oxytocin (pEC50 = 4.29 ± 0.17 and Emax = 86.69 ± 1.56% at 10-3 M) contractions. The effect of ( -)-carvone was altered in the presence of 4-aminopyridine, glibenclamide, L-NAME, or methylene blue. Mice pre-treated with (-)-carvone at a dose of 100 mg/kg showed a significant reduction in the number of writhing after oxytocin administration. No toxicity was observed after oral administration of 1 g/kg ( -)-carvone. Taken together, we showed that (-)-carvone reduced writhing by a spasmolytic effect, probably through the participation of KV and KATP channels and the nitric oxide pathway.

The anion exchanger slc26a3 regulates colonic mucus expansion during steady state and in response to prostaglandin E2, while Cftr regulates de novo mucus release in response to carbamylcholine.

The intestinal epithelium is covered by mucus that protects the tissue from the luminal content. Studies have shown that anion secretion via the cystic fibrosis conductance regulator (Cftr) regulates mucus formation in the small intestine. However, mechanisms regulating mucus formation in the colon are less understood. The aim of this study was to explore the role of anion transport in the regulation of mucus formation during steady state and in response to carbamylcholine (CCh) and prostaglandin E2 (PGE2). The broad-spectrum anion transport inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS), CftrdF508 (CF) mice, and the slc26a3 inhibitor SLC26A3-IN-2 were used to inhibit anion transport. In the distal colon, steady-state mucus expansion was reduced by SLC26A3-IN-2 and normal in CF mice. PGE2 stimulated mucus expansion without de novo mucus release in wild type (WT) and CF colon via slc26a3 sensitive mechanisms, while CCh induced de novo mucus secretion in WT but not in CF colon. However, when added simultaneously, CCh and PGE2 stimulated de novo mucus secretion in the CF colon via DIDS-sensitive pathways. A similar response was observed in CF ileum that responded to CCh and PGE2 with DIDS-sensitive de novo mucus secretion. In conclusion, this study suggests that slc26a3 regulates colonic mucus expansion, while Cftr regulates CCh-induced de novo mucus secretion from ileal and distal colon crypts. Furthermore, these findings demonstrate that in the absence of a functional Cftr channel, parallel stimulation with CCh and PGE2 activates additional anion transport processes that help release mucus from intestinal goblet cells.

17ß-estradiol induces hyperresponsiveness in guinea pig airway smooth muscle by inhibiting the plasma membrane Ca2+-ATPase.

High serum estrogen concentrations are associated with asthma development and severity, suggesting a link between estradiol and airway hyperresponsiveness (AHR). 17ß-estradiol (E2) has non-genomic effects via Ca2+ regulatory mechanisms; however, its effect on the plasma membrane Ca2+-ATPases (PMCA1 and 4) and sarcoplasmic reticulum Ca2+-ATPase (SERCA) is unknown. Hence, in the present study, we aim to demonstrate if E2 favors AHR by increasing intracellular Ca2+ concentrations in guinea pig airway smooth muscle (ASM) through a mechanism involving Ca2+-ATPases. In guinea pig ASM, Ca2+ microfluorometry, muscle contraction, and Western blot were evaluated. Then, we performed molecular docking analysis between the estrogens and Ca2+ ATPases. In tracheal rings, E2 produced AHR to carbachol. In guinea pig myocytes, acute exposure to physiological levels of E2 modified the transient Ca2+ peak induced by caffeine to a Ca2+ plateau. The incubation with PMCA inhibitors (lanthanum and carboxyeosin, CE) partially reversed the E2-induced sustained plateau in the caffeine response. In contrast, cyclopiazonic acid (SERCA inhibitor), U-0126 (an inhibitor of ERK 1/2), and choline chloride did not modify the Ca2+ plateau produced by E2. The mitochondrial uniporter activity and the capacitative Ca2+ entry were unaffected by E2. In guinea pig ASM, Western blot analysis demonstrated PMCA1 and PMCA4 expression. The results from the docking modeling demonstrate that E2 binds to both plasma membrane ATPases. In guinea pig tracheal smooth muscle, inhibiting the PMCA with CE, induced hyperresponsiveness to carbachol. 17ß-estradiol produces hyperresponsiveness by inhibiting the PMCA in the ASM and could be one of the mechanisms responsible for the increase in asthmatic crisis in women.

Further investigations on the influence of protein phosphatases on the signaling of muscarinic receptors in the atria of mouse hearts.

The vagal regulation of cardiac function involves acetylcholine (ACh) receptor activation followed by negative chronotropic and negative as well as positive inotropic effects. The resulting signaling pathways may include Gi/o protein-coupled reduction in adenylyl cyclase (AC) activity, direct Gi/o protein-coupled activation of ACh-activated potassium current (IKACh), inhibition of L-type calcium ion channels, and/or the activation of protein phosphatases. Here, we studied the role of the protein phosphatases 1 (PP1) and 2A (PP2A) for muscarinic receptor signaling in isolated atrial preparations of transgenic mice with cardiomyocyte-specific overexpression of either the catalytic subunit of PP2A (PP2A-TG) or the inhibitor-2 (I2) of PP1 (I2-TG) or in double transgenic mice overexpressing both PP2A and I2 (DT). In mouse left atrial preparations, carbachol (CCh), cumulatively applied (1 nM-10 µM), exerted at low concentrations a negative inotropic effect followed by a positive inotropic effect at higher concentrations. This biphasic effect was noted with CCh alone as well as when CCh was added after ß-adrenergic pre-stimulation with isoprenaline (1 µM). Whereas the response to stimulation of ß-adrenoceptors or adenosine receptors (used as controls) was changed in PP2A-TG, the response to CCh was unaffected in atrial preparations from all transgenic models studied here. Therefore, the present data tentatively indicate that neither PP2A nor PP1, but possibly other protein phosphatases, is involved in the muscarinic receptor-induced inotropic and chronotropic effects in the mouse heart.

Cholinergic Control of GnRH Neuron Physiology and Luteinizing Hormone Secretion in Male Mice: Involvement of ACh/GABA Cotransmission.

Gonadotropin-releasing hormone (GnRH)-synthesizing neurons orchestrate reproduction centrally. Early studies have proposed the contribution of acetylcholine (ACh) to hypothalamic control of reproduction, although the causal mechanisms have not been clarified. Here, we report that in vivo pharmacogenetic activation of the cholinergic system increased the secretion of luteinizing hormone (LH) in orchidectomized mice. 3DISCO immunocytochemistry and electron microscopy revealed the innervation of GnRH neurons by cholinergic axons. Retrograde viral labeling initiated from GnRH-Cre neurons identified the medial septum and the diagonal band of Broca as exclusive sites of origin for cholinergic afferents of GnRH neurons. In acute brain slices, ACh and carbachol evoked a biphasic effect on the firing rate in GnRH neurons, first increasing and then diminishing it. In the presence of tetrodotoxin, carbachol induced an inward current, followed by a decline in the frequency of miniature postsynaptic currents (mPSCs), indicating a direct influence on GnRH cells. RT-PCR and whole-cell patch-clamp studies revealed that GnRH neurons expressed both nicotinic (α4ß2, α3ß4, and α7) and muscarinic (M1-M5) AChRs. The nicotinic AChRs contributed to the nicotine-elicited inward current and the rise in firing rate. Muscarine via M1 and M3 receptors increased, while via M2 and M4 reduced the frequency of both mPSCs and firing. Optogenetic activation of channelrhodopsin-2-tagged cholinergic axons modified GnRH neuronal activity and evoked cotransmission of ACh and GABA from a subpopulation of boutons. These findings confirm that the central cholinergic system regulates GnRH neurons and activates the pituitary-gonadal axis via ACh and ACh/GABA neurotransmissions in male mice.

IP3RPEP6, a novel peptide inhibitor of IP3 receptor channels that does not affect connexin-43 hemichannels.

AIM: Inositol 1,4,5-trisphosphate receptors (IP3 Rs) are intracellular Ca2+ -release channels with crucial roles in cell function. Current IP3 R inhibitors suffer from off-target effects and poor selectivity towards the three distinct IP3 R subtypes. We developed a novel peptide inhibitor of IP3 Rs and determined its effect on connexin-43 (Cx43) hemichannels, which are co-activated by IP3 R stimulation. METHODS: IP3RPEP6 was developed by in silico molecular docking studies and characterized by on-nucleus patch-clamp experiments of IP3 R2 channels and carbachol-induced IP3 -mediated Ca2+ responses in IP3 R1, 2 or 3 expressing cells, triple IP3 R KO cells and astrocytes. Cx43 hemichannels were studied by patch-clamp and ATP-release approaches, and by inhibition with Gap19 peptide. IP3RPEP6 interactions with IP3 Rs were verified by co-immunoprecipitation and affinity pull-down assays. RESULTS: IP3RPEP6 concentration-dependently reduced the open probability of IP3 R2 channels and competitively inhibited IP3 Rs in an IC50 order of IP3 R2 (~3.9 µM) < IP3 R3 (~4.3 µM) < IP3 R1 (~9.0 µM), without affecting Cx43 hemichannels or ryanodine receptors. IP3RPEP6 co-immunoprecipitated with IP3 R2 but not with IP3 R1; interaction with IP3 R3 varied between cell types. The IC50 of IP3RPEP6 inhibition of carbachol-induced Ca2+ responses decreased with increasing cellular Cx43 expression. Moreover, Gap19-inhibition of Cx43 hemichannels significantly reduced the amplitude of the IP3 -Ca2+ responses and strongly increased the EC50 of these responses. Finally, we identified palmitoyl-8G-IP3RPEP6 as a membrane-permeable IP3RPEP6 version allowing extracellular application of the IP3 R-inhibiting peptide. CONCLUSION: IP3RPEP6 inhibits IP3 R2/R3 at concentrations that have limited effects on IP3 R1. IP3 R activation triggers hemichannel opening, which strongly affects the amplitude and concentration-dependence of IP3 -triggered Ca2+ responses.

Cantharidin and sodium fluoride attenuate the negative inotropic effects of carbachol in the isolated human atrium.

Carbachol, an agonist at muscarinic receptors, exerts a negative inotropic effect in human atrium. Carbachol can activate protein phosphatases (PP1 or PP2A). We hypothesized that cantharidin or sodium fluoride, inhibitors of PP1 and PP2A, may attenuate a negative inotropic effect of carbachol. During bypass-surgery trabeculae carneae of human atrial preparations (HAP) were obtained. These trabeculae were mounted in organ baths and electrically stimulated (1 Hz). Force of contraction was measured under isometric conditions. For comparison, we studied isolated electrically stimulated left atrial preparations (LA) from mice. Cantharidin (100 µM) and sodium fluoride (3 mM) increased force of contraction in LA (n = 5-8, p < 0.05) by 113% ± 24.5% and by 100% ± 38.2% and in HAP (n = 13-15, p < 0.05) by 625% ± 169% and by 196% ± 23.5%, respectively. Carbachol (1 µM) alone exerted a rapid transient maximum negative inotropic effect in LA (n = 6) and HAP (n = 14) to 46.9% ± 3.63% and 19.4% ± 3.74%, respectively (p < 0.05). These negative inotropic effects were smaller in LA (n = 4-6) and HAP (n = 9-12) pretreated with 100 µM cantharidin and amounted to 58.0% ± 2.27% and 59.2% ± 6.19% or 3 mM sodium fluoride to 63.7% ± 9.84% and 46.3% ± 5.69%, (p < 0.05). We suggest that carbachol, at least in part, exerts a negative inotropic effect in the human atrium by stimulating the enzymatic activity of PP1 and/or PP2A.

Excitatory purinergic and cholinergic expression changed in a partial bladder outlet obstruction-induced overactive bladder rat model.

Overactive bladder (OAB) is a common, long-term symptom complex with a high prevalence in women worldwide. OAB has caused a social burden, and effective treatments are urgently needed. However, the pathogenesis of OAB has yet to be elucidated. Model rats underwent bladder outlet obstruction surgery. In the 2nd, 3rd, and 4th weeks after surgery, metabolic cages were used to detect the 12 h urine volume of rats in the sham and model groups. The urodynamic parameters bladder leak point pressure (BPLL), maximum voiding pressure (MVP), residual volume (RV), maximum bladder capacity (MBC), bladder compliance (BC), voided efficiency (VE), and non-voiding contractions (NVCs) were also detected. Moreover, the contractile responses of isolated detrusor muscles to electrical and carbachol stimulation were examined at the abovementioned time points. At the 4th week after surgery, the bladders of both groups were obtained for hematoxylin-eosin (H&E) and Masson's trichrome staining. Real-time qPCR and Western blot were performed to quantify the expression of choline acetyltransferase (ChAT) and solute carrier family 17 member 9 (SLC17A9). At week 4, compared with the sham group, the 12 h urine volume of PBOO group increased significantly. The BLPP, MVP, VE, MBC, and NVCs increased significantly, and the VE was significantly reduced in 4-week PBOO group. The contractile responses of isolated detrusor muscles to electrical and carbachol stimulation significantly increased in 4-week PBOO group. In the 4-week PBOO group, the bladder wall and the ratio of bladder muscle to collagen within the bladder smooth muscle layer wall were significantly higher than those in the sham group. ChAT and SLC17A9 mRNA and protein expression in the OAB model rats significantly increased. At 4 weeks after PBOO, the OAB model was successfully established. The gene and protein expression levels of ChAT and SLC17A9 increased in the bladder of the OAB model, suggesting that OAB may be related to increased excitatory purinergic and cholinergic expression.

Comparative Assessment of Disturbances of Contractions of the Isolated Uterus in 3- and 9-Month-Old Rats with a Model of Autism.

We performed a comparative study of the effects of carbachol, α,ß-methylene-ATP, ß,γ-methylene-ATP, and electric field stimulation on the contractile activity of the isolated uterus from rats aged 3 and 9 months with valproic model of autism. The contractile responses of isolated rat uterine preparations induced by P2X-receptor agonists α,ß-methylene-ATP and ß,γ-methylene-ATP were significantly lower than in the control. In addition, the contractions of the isolated uterus of 9-month-old rats induced by carbachol were significantly lower than in controls. No significant differences in uterine smooth muscle contractions in both age groups of rats induced by electric field stimulation in comparison with the control were found. Thus, significant impairment of uterine contractile activity was revealed in rats with valproic model of autism, which persisted up to the age of 9 months. The absence of changes in the contractions induced by electric field stimulation suggests that the changes in the contractile activity of the uterus of the rats with modeled autism spectrum disorder are caused by the disorders occurring at the postsynaptic level.

The role of rhoA/rho-kinase and PKC in the inhibitory effect of L-cysteine/H2S pathway on the carbachol-mediated contraction of mouse bladder smooth muscle.

We investigated the role of RhoA/Rho-kinase (ROCK) and PKC in the inhibitory effect of L-cysteine/hydrogen sulfide (H2S) pathway on the carbachol-mediated contraction of mouse bladder smooth muscle. Carbachol (10-8-10-4 M) induced a concentration-dependent contraction in bladder tissues. L-cysteine (H2S precursor; 10-2 M) and exogenous H2S (NaHS; 10-3 M) reduced the contractions evoked by carbachol by ~ 49 and ~ 53%, respectively, relative to control. The inhibitory effect of L-cysteine on contractions to carbachol was reversed by 10-2 M PAG (~ 40%) and 10-3 M AOAA (~ 55%), cystathionine-gamma-lyase (CSE) and cystathionine-ß-synthase (CBS) inhibitor, respectively. Y-27632 (10-6 M) and GF 109203X (10-6 M), a specific ROCK and PKC inhibitor, respectively, reduced contractions evoked by carbachol (~ 18 and ~ 24% respectively), and the inhibitory effect of Y-27632 and GF 109203X on contractions was reversed by PAG (~ 29 and ~ 19%, respectively) but not by AOAA. Also, Y-27632 and GF 109203X reduced the inhibitory responses of L-cysteine on the carbachol-induced contractions (~ 38 and ~ 52% respectively), and PAG abolished the inhibitory effect of L-cysteine on the contractions in the presence of Y-27632 (~ 38%). Also, the protein expressions of CSE, CBS, and 3-MST enzymes responsible for endogenous H2S synthesis were detected by Western blot method. H2S level was increased by L-cysteine, Y-27632, and GF 109203X (from 0.12 ± 0.02 to 0.47 ± 0.13, 0.26 ± 0.03, and 0.23 ± 0.06 nmol/mg respectively), and this augmentation in H2S level decreased with PAG (0.17 ± 0.02, 0.15 ± 0.03, and 0.07 ± 0.04 nmol/mg respectively). Furthermore, L-cysteine and NaHS reduced carbachol-induced ROCK-1, pMYPT1, and pMLC20 levels. Inhibitory effects of L-cysteine on ROCK-1, pMYPT1, and pMLC20 levels, but not of NaHS, were reversed by PAG. These results suggest that there is an interaction between L-cysteine/H2S and RhoA/ROCK pathway via inhibition of ROCK-1, pMYPT1, and pMLC20, and the inhibition of RhoA/ROCK and/or PKC signal pathway may be mediated by the CSE-generated H2S in mouse bladder.

Evaluation of Disorders of Contractile Activity of Smooth Muscle Tissues in 9-Month-Old Rats with a Model of Autism.

We studied the effect of carbachol, P2 receptor agonists, and electric field stimulation on mechanical activity of isolated preparations of the duodenum, ileum, bladder, and vas deferens in 9-month-old rats with the valproic acid-induced model of autism. It was found that the contractions and relaxations of the isolated intestine of the experimental rats caused by various agonists and electric field stimulation did not differ from those in control animals. Carbachol induced more significant contractions of the bladder in rats with autism model than in controls and electric field stimulation induced more significant contractions of the vas deferens. We did not find significant differences in the effect of ATP, α,ß-methylene-ATP and 2-methylthio-ATP on the contraction and relaxation of the studied smooth muscle organs of rats of the experimental and control groups. It was concluded that 9-month-old rats with an autism model retained increased contractile activity of the bladder and vas deferens, while intestinal contractions in experimental and control animals of this age did not differ significantly.

Starving a Cell Promotes Airway Smooth Muscle Relaxation: Inhibition of Glycolysis Attenuates Excitation-Contraction Coupling.

Bronchomotor tone modulated by airway smooth muscle shortening represents a key mechanism that increases airway resistance in asthma. Altered glucose metabolism in inflammatory and airway structural cells is associated with asthma. Although these observations suggest a causal link between glucose metabolism and airway hyperresponsiveness, the mechanisms are unclear. We hypothesized that glycolysis modulates excitation-contraction coupling in human airway smooth muscle (HASM) cells. Cultured HASM cells from human lung donors were subject to metabolic screenings using Seahorse XF cell assay. HASM cell monolayers were treated with vehicle or PFK15 (1-(Pyridin-4-yl)-3-(quinolin-2-yl)prop-2-en-1-one), an inhibitor of PFKFB3 (PFK-1,6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3) that generates an allosteric activator for glycolysis rate-limiting enzyme PFK1 (phosphofructokinase 1), for 5-240 minutes, and baseline and agonist-induced phosphorylation of MLC (myosin light chain), MYPT1 (myosin phosphatase regulatory subunit 1), Akt, RhoA, and cytosolic Ca2+ were determined. PFK15 effects on metabolic activity and contractile agonist-induced bronchoconstriction were determined in human precision-cut lung slices. Inhibition of glycolysis attenuated carbachol-induced excitation-contraction coupling in HASM cells. ATP production and bronchodilator-induced cAMP concentrations were also attenuated by glycolysis inhibition in HASM cells. In human small airways, glycolysis inhibition decreased mitochondrial respiration and ATP production and attenuated carbachol-induced bronchoconstriction. The findings suggest that energy depletion resulting from glycolysis inhibition is a novel strategy for ameliorating HASM cell shortening and bronchoprotection of human small airways.

Cysteinyl-maresin 3 inhibits IL-13 induced airway hyperresponsiveness through alternative activation of the CysLT1 receptor.

BACKGROUND: Cysteinyl-maresins, also known as maresin-conjugates in tissue regeneration (MCTRs), are recently discovered lipid mediators proposed to reduce airway inflammation. OBJECTIVE: To investigate the influence of MCTRs on IL-13-induced airway hyperresponsiveness in isolated human and mice airways. METHODS: Before responsiveness to contractile agonists were assessed in myographs, human small bronchi were cultured for 2 days and mouse tracheas were cultured for 1-4 days. During the culture procedure airways were exposed to interleukin (IL)-13 in the presence or absence of MCTRs. Signalling mechanisms were explored using pharmacologic agonists and antagonists, and genetically modified mice. RESULTS: IL-13 treatment increased contractions to histamine, carbachol and leukotriene D4 (LTD4) in human small bronchi, and to 5-hydroxytryptamine (5-HT) in mouse trachea. In both preparations, co-incubation of the explanted tissues with MCTR3 reduced the IL-13 induced enhancement of contractions. In mouse trachea, this inhibitory effect of MCTR3 was blocked by three different CysLT1 receptor antagonists (montelukast, zafirlukast and pobilukast) during IL-13 exposure. Likewise, MCTR3 failed to reduce the IL-13-induced 5-HT responsiveness in mice deficient of the CysLT1 receptor. However, co-incubation with the classical CysLT1 receptor agonist LTD4 did not alter the IL-13-induced 5-HT hyperreactivity. CONCLUSIONS: MCTR3, but not LTD4, decreased the IL-13-induced airway hyperresponsiveness by activation of the CysLT1 receptor. The distinct actions of the two lipid mediators on the CysLT1 receptor suggest an alternative signalling pathway appearing under inflammatory conditions, where this new action of MCTR3 implicates potential to inhibit airway hyperresponsiveness in asthma.

Co-administration of sodium hydrosulfide and tadalafil modulates hypoxia and oxidative stress on bladder dysfunction in a rat model of bladder outlet obstruction.

PURPOSE: This study aimed to assess the possible healing effect of combination treatment with a hydrogen sulfide (H2S) donor, sodium hydrosulfide (NaHS) plus tadalafil on partial bladder outlet obstruction (PBOO)-induced bladder dysfunction. MATERIALS AND METHODS: A total of 75 male Sprague-Dawley rats aged 10-wk and 300-350g were divided into five groups; control; PBOO; PBOO+NaHS (5.6mg/kg/day, i.p., 6-wk); PBOO+tadalafil (2mg/kg/day, oral, 6-wk) and PBOO+NaHS+tadalafil. PBOO was created by partial urethral ligation. 6 weeks after obstruction, the in vitro contractile responses of the detrusor muscle and Western blotting, H2S and malondialdehyde assay were performed in bladder tissues. RESULTS: There was an increase in bladder weight(p<0.001) and a decrease in contractile responses to KCL(p<0.001), carbachol(p<0.01), electrical field stimulation(p<0.05) and ATP (p<0.001) in the detrusor smooth muscle of obstructed rats which was normalized after the combination treatment. Cystathionine γ-lyase and cystathionine ß-synthase, and nuclear factor kappa B protein levels did not significantly differ among groups. The obstruction induced decrement in 3-mercaptopyruvate sulfur transferase protein expression(p<0.001) and H2S levels(p<0.01) as well as increment in protein expressions of neuronal nitric oxide synthase (NO, p<0.001), endothelial NOS (p<0.05), inducible NOS(p<0.001), hypoxia-inducible factor 1-alpha (p<0.01), and malondialdehyde levels (p<0.01), when combined treatment entirely normalized. CONCLUSIONS: Combination therapy has beneficial effects on bladder dysfunction via regulating both H2S and nitric oxide pathways as well as downregulation of oxidative stress and hypoxia. The synergistic effect of H2S and nitric oxide is likely to modulate bladder function, which supports the combined therapy for enhancing clinical outcomes in men with BPH/LUTS.

Cholinergic signaling impairs cardiomyocyte cohesion.

AIM: Cardiac autonomic nervous system (ANS) dysregulation is a hallmark of several cardiovascular diseases. Adrenergic signaling enhanced cardiomyocyte cohesion via PKA-mediated plakoglobin phosphorylation at serine 665, referred to as positive adhesiotropy. This study investigated cholinergic regulation of cardiomyocyte cohesion using muscarinic receptor agonist carbachol (CCH). METHODS: Dissociation assays, Western blot analysis, immunostaining, atomic force microscopy (AFM), immunoprecipitation, transmission electron microscopy (TEM), triton assays, and siRNA knockdown of genes were performed in either HL-1 cells or plakoglobin (PG) wild type (Jup+/+ ) and knockout (Jup-/- ) mice, which served as a model for arrhythmogenic cardiomyopathy. RESULTS: In HL-1 cells grown in norepinephrine (NE)-containing medium for baseline adrenergic stimulation, and murine cardiac slice cultures from Jup+/+ and Jup-/- mice CCH treatment impaired cardiomyocyte cohesion. Immunostainings and AFM experiments revealed that CCH reduced desmoglein 2 (DSG2) localization and binding at cell borders. Furthermore, CCH reduced intercalated disc plaque thickness in both Jup+/+ and Jup-/- mice, evidenced by TEM analysis. Immunoprecipitation experiments in HL-1 cells revealed no changes in DSG2 interaction with desmoplakin (DP), plakophilin 2 (PKP2), PG, and desmin (DES) after CCH treatment. However, knockdown of any of the above proteins abolished CCH-mediated loss of cardiomyocyte cohesion. Furthermore, in HL-1 cells, CCH inhibited adrenergic-stimulated ERK phosphorylation but not PG phosphorylation at serine 665. In addition, CCH activated the AKT/GSK-3ß axis in the presence of NE. CONCLUSION: Our results demonstrate that cholinergic signaling antagonizes the positive effect of adrenergic signaling on cardiomyocyte cohesion and thus causes negative adhesiotropy independent of PG phosphorylation.

Effect of Intracameral Carbachol and Epinephrine Use on Choroidal Thickness After Uncomplicated Phacoemulsification.

BACKGROUND Safety concerns about drugs used intracamerally during cataract surgery have been the subject of many studies. In this study, the effect of using intracameral carbachol and epinephrine on choroidal thickness was evaluated. MATERIAL AND METHODS This prospective interventional study included 81 eyes of 81 patients undergoing cataract surgery. During cataract surgery, intracameral carbachol was administered to 27 eyes, intracameral epinephrine was administered to 20 eyes, and 34 eyes were the control group. Macular choroidal thickness measurement was performed before, 1 day, and 1 week after phacoemulsification surgery in all patients using optical coherence tomography. RESULTS Subfoveal choroidal thickness was significantly reduced at day 1 and week 1 in the group receiving intraoperative carbachol compared with preoperative measurement (P=0.016). In addition, choroidal thickness in the 500 µm nasal fovea was significantly reduced in the carbachol group at 1st week compared to the preoperative measurement (P=0.008). There was no significant difference in postoperative subfoveal thickness in the intraoperative epinephrine group and control group (P=0.179 and P=0.953, respectively). Choroidal thickness at 1000 µm nasal fovea was significantly higher in the epinephrine group at postoperative 1st day than preoperative and postoperative 1st week values (P=0.009). CONCLUSIONS The use of intracameral epinephrine caused an increase in choroidal thickness 1000 µm nasal of the fovea, while intracameral carbachol caused thinning in the subfoveal and 500 µm nasal quadrant. Intracameral drug administration during cataract surgery may be associated with posterior segment complications.

Inhibition of Full Smooth Muscle Contraction in Isolated Human Detrusor Tissues by Mirabegron Is Limited to Off-Target Inhibition of Neurogenic Contractions.

Mirabegron is used for treatment of storage symptoms in overactive bladder (OAB) caused by spontaneous bladder smooth muscle contractions. However, owing to limitations in available studies using human tissues, central questions are still unresolved, including mechanisms underlying improvements by mirabegron and its anticontractile effects in the detrusor. Here, we assessed concentration-dependent mirabegron effects on contractions of human detrusor tissues in frequency-response curves and concentration-response curves for different cholinergic and noncholinergic agonists. Detrusor tissues were sampled from patients undergoing radical cystectomy. Contractions were induced by electric field stimulation (EFS) and by cumulative concentrations of cholinergic agonists, endothelin-1, and the thromboxane A2 analog U46619. EFS-induced contractions were inhibited using 10 µM mirabegron, but not using 1 µM. Inhibition by 10 µM mirabegron was resistant to the ß 3-adrenergic antagonist L-748,337. Concentration-dependent contractions by carbachol were not inhibited by 1 µM or 10 µM mirabegron. Concentration-response curves for methacholine were slightly right-shifted by 10 µM, but not 1 µM mirabegron. Concentration-dependent contractions by endothelin-1 or U46619 were not changed by mirabegron. In contrast, the muscarinic antagonist tolterodine right-shifted concentration-response curves for carbachol and methacholine and inhibited EFS-induced contractions. In conclusion, inhibition of neurogenic contractions in isolated detrusor tissues by mirabegron requires concentrations highly exceeding known plasma levels during standard dosing and the known binding constant (Ki values) for ß 3-adrenoceptors. Full contractions by cholinergic agonists, endothelin-1, and U46619 are not affected by therapeutic concentrations of mirabegron. Improvements of storage symptoms are most likely not imparted by inhibition of ß 3-adrenoceptors in the bladder wall itself. SIGNIFICANCE STATEMENT: Mirabegron is used for overactive bladder (OAB) treatment, but the underlying mechanisms are unclear, and preclinical and clinical findings are controversial due to limitations in available studies. Our findings suggest that inhibition of detrusor contractions by mirabegron is limited to neurogenic contractions, which requires unphysiologic concentrations and does not involve ß 3-adrenoceptors. Mechanisms accounting for improvements of OAB by mirabegron are located outside the urinary bladder.

Lippia origanoides essential oil induces tocolytic effect in virgin rat uterus and inhibits writhing in a dysmenorrhea mouse model.

ETHNOPHARMACOLOGICAL RELEVANCE: The species Lippia origanoides Kunth, popularly known as "salva-de-marajó", is used in Brazilian traditional "quilombola" communities to treat menstrual cramps and uterine inflammation. AIM OF THE STUDY: Evaluate the spasmolytic activity of Lippia origanoides essential oil (LOO) on experimental models of uterine conditions related to menstrual cramps and investigate its mechanism of action. MATERIALS AND METHODS: Virgin rat-isolated uterus was mounted in the organ bath apparatus to evaluate the spasmolytic effect of LOO on basal tonus and contractions induced by carbachol, KCl, or oxytocin. We used pharmacological agents to verify the relaxation mechanism of LOO. The evaluation of uterine contractility in virgin rats, after treatment with LOO for three consecutive days, was carried out by the construction of a concentration-response curve with oxytocin or carbachol. The primary dysmenorrhea animal model was replicated with an injection of estradiol cypionate in female mice for three consecutive days, followed by intraperitoneal application of oxytocin. RESULTS: LOO relaxed the rat uterus precontracted with 10-2 IU/mL oxytocin (logEC50 = 1.98 ± 0.07), 1 µM carbachol (logEC50 = 1.42 ± 0.07) or 60 mM KCl (logEC50 = 1.53 ± 0.05). It was also able relax uterus on spontaneous contractions (logEC50 = 0.41 ± 0.05). Preincubation with glibenclamide, propranolol, phentolamine or L-NAME in contractions induced by carbachol did not alter significantly the relaxing effect of LOO. However, in the presence of 4-aminopyridine, CsCl or tetraethylammonium there was a reduction of LOO potency, whereas the blockers methylene blue, ODQ, aminophylline and heparin potentiated the LOO relaxing effect. Preincubation with LOO in a Ca2+ free medium at concentrations of 27 µg/mL or 81 µg/mL reduced the contraction induced by carbachol. The administration of LOO for 3 days did not alter uterus contractility. The treatment with LOO at 30 or 100 mg/kg intraperitoneally, or 100 mg/kg orally, inhibited writhing in female mice. The association of LOO at 10 mg/kg with nifedipine or mefenamic acid potentiated writhing inhibition in mice. CONCLUSIONS: The essential oil of L. origanoides has tocolytic activity in rat isolated uterus pre-contracted with KCl, oxytocin, or carbachol. This effect is possibly related to the opening of potassium channels (Kir, KV, and KCa), cAMP increase, and diminution of intracellular Ca2+. This relaxant effect, probably, contributed to reduce the number of writhings in an animal model of dysmenorrhea being potentiated by nifedipine or mefenamic acid. Taken together, the results here presented indicate that this species has a pharmacological potential for the treatment of primary dysmenorrhea, supporting its use in folk medicine.

Cross-talk of cholinergic and ß-adrenergic receptor signalling in chronic myeloid leukemia K562 cells.

In many studies on breast, skin and intestinal cancers, ß-adrenergic receptor antagonists have been shown to inhibit cell proliferation and angiogenesis and increase apoptosis in cancers. Carbachol inhibits chronic myeloid leukaemia K562 cell proliferation. Beta-blockers are known to inhibit cell progression. The aim of this study is to explain the mechanism of action of ß-adrenergic receptors agonists and antagonists on apoptosis in chronic myeloid leukaemia cells. We tried to determine the effect of combined treatment of ß-adrenergic and cholinergic drugs on adrenergic ß1 and ß2 gene expression, cell proliferation and apoptosis in chronic myeloid leukaemia K562 cells. Cell proliferation was evaluated by the 5-bromo-2-deoxy-uridine (BrdU) incorporation kit. Caspase 3, 8, 9 activities were measured by the caspase assay kit. Protein expression level was detected by western blotting. We found that exposure to propranolol either by combination with carbachol facilitates additive effects on inhibition of caspase 3 and 8 expression in chronic myeloid leukaemia K562 cells. However, caspase 9 expression level was increased by propranolol alone or with propranolol and carbachol combination. The combined therapy of cholinergic and adrenergic receptor drugs will decrease cell proliferation in K562 cells. This decrease in cell proliferation may be mediated by the mitochondrial-dependent intrinsic apoptosis pathway.