Local carbachol application induces oral microvascular recruitment and improves gastric tissue oxygenation during hemorrhagic shock in dogs.

Introduction: Hemorrhagic shock is characterized by derangements of the gastrointestinal microcirculation. Topical therapy with nitroglycerine or iloprost improves gastric tissue oxygenation but not regional perfusion, probably due to precapillary adrenergic innervation. Therefore, this study was designed to investigate the local effect of the parasympathomimetic carbachol alone and in combination with either nitroglycerine or iloprost on gastric and oral microcirculation during hemorrhagic shock. Methods: In a cross-over design five female foxhounds were repeatedly randomized into six experimental groups. Carbachol, or carbachol in combination with either nitroglycerine or iloprost were applied topically to the oral and gastric mucosa. Saline, nitroglycerine, or iloprost application alone served as control groups. Then, a fixed-volume hemorrhage was induced by arterial blood withdrawal followed by blood retransfusion after 1h of shock. Gastric and oral microcirculation was determined using reflectance spectrophotometry and laser Doppler flowmetry. Oral microcirculation was visualized with videomicroscopy. Statistics: 2-way-ANOVA for repeated measurements and Bonferroni post-hoc analysis (mean ± SEM; p < 0.05). Results: The induction of hemorrhage led to a decrease of gastric and oral tissue oxygenation, that was ameliorated by local carbachol and nitroglycerine application at the gastric mucosa. The sole use of local iloprost did not improve gastric tissue oxygenation but could be supplemented by local carbachol treatment. Adding carbachol to nitroglycerine did not further increase gastric tissue oxygenation. Gastric microvascular blood flow remained unchanged in all experimental groups. Oral microvascular blood flow, microvascular flow index and total vessel density decreased during shock. Local carbachol supply improved oral vessel density during shock and oral microvascular flow index in the late course of hemorrhage. Conclusion: The specific effect of shifting the autonomous balance by local carbachol treatment on microcirculatory variables varies between parts of the gastrointestinal tract. Contrary to our expectations, the improvement of gastric tissue oxygenation by local carbachol or nitroglycerine application was not related to increased microvascular perfusion. When carbachol is used in combination with local vasodilators, the additional effect on gastric tissue oxygenation depends on the specific drug combination. Therefore, modulation of tissue oxygen consumption, mitochondrial function or alterations in regional blood flow distribution should be investigated.

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.

Lipopolysaccharide-induced sepsis impairs M2R-GIRK signaling in the mouse sinoatrial node.

Sepsis has emerged as a global health burden associated with multiple organ dysfunction and 20% mortality rate in patients. Numerous clinical studies over the past two decades have correlated the disease severity and mortality in septic patients with impaired heart rate variability (HRV), as a consequence of impaired chronotropic response of sinoatrial node (SAN) pacemaker activity to vagal/parasympathetic stimulation. However, the molecular mechanism(s) downstream to parasympathetic inputs have not been investigated yet in sepsis, particularly in the SAN. Based on electrocardiography, fluorescence Ca2+ imaging, electrophysiology, and protein assays from organ to subcellular level, we report that impaired muscarinic receptor subtype 2-G protein-activated inwardly-rectifying potassium channel (M2R-GIRK) signaling in a lipopolysaccharide-induced proxy septic mouse model plays a critical role in SAN pacemaking and HRV. The parasympathetic responses to a muscarinic agonist, namely IKACh activation in SAN cells, reduction in Ca2+ mobilization of SAN tissues, lowering of heart rate and increase in HRV, were profoundly attenuated upon lipopolysaccharide-induced sepsis. These functional alterations manifested as a direct consequence of reduced expression of key ion-channel components (GIRK1, GIRK4, and M2R) in the mouse SAN tissues and cells, which was further evident in the human right atrial appendages of septic patients and likely not mediated by the common proinflammatory cytokines elevated in sepsis.

Carbachol, along with calcium, indicates new strategy in neural differentiation of human adipose tissue-derived mesenchymal stem cells in vitro.

Introduction: Over the past few years, stem cells have represented a promising treatment in neurological disorders due to the well-defined characteristics of their capability to proliferate and differentiate into any cell type, both in vitro and in vivo. Additionally, previous studies have shown that calcium signaling modulates the proliferation and differentiation of neural progenitor cells. The present study investigated the effect of carbachol (CCh), a cholinergic agonist activating acetylcholine receptors, with and without calcium, on the neural differentiation of human adipose tissue-derived mesenchymal stem cells (hADSCs) in neural media, including forskolin and 3-isobutyl-1-methyl-xanthine and retinoic acid. Methods: For this purpose, first, the MTT assay and acridine orange staining were studied to obtain the optimal concentration of CCh. Next, the differentiation tests, such as cellular calcium assay as well as evaluation of qualitative and quantitative expression of neuronal index markers through immunofluorescence staining and gene expression analysis, respectively, were performed on days 7 and 14 of the differentiation period. Results: According to the results, CCh at 1 µM concentration had no cytotoxicity on hADSCs and also induced cell proliferation. Furthermore, CCh with and without calcium increased the expression of neural-specific genes (NSE, MAP2, ß-III-tubulin, and MAPK3) and proteins (γ-enolase, MAP2, and ß-III-tubulin) as well as the amount of calcium in differentiated hADSCs at 7 and 14 days after induction. Conclusions: In conclusion, the findings suggest that CCh acts as an influential therapeutic factor in the field of neural regenerative medicine and research.

PITX2 Knockout Induces Key Findings of Electrical Remodeling as Seen in Persistent Atrial Fibrillation.

BACKGROUND: Electrical remodeling in human persistent atrial fibrillation is believed to result from rapid electrical activation of the atria, but underlying genetic causes may contribute. Indeed, common gene variants in an enhancer region close to PITX2 (paired-like homeodomain transcription factor 2) are strongly associated with atrial fibrillation, but the mechanism behind this association remains unknown. This study evaluated the consequences of PITX2 deletion (PITX2-/-) in human induced pluripotent stem cell-derived atrial cardiomyocytes. METHODS: CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) was used to delete PITX2 in a healthy human iPSC line that served as isogenic control. Human induced pluripotent stem cell-derived atrial cardiomyocytes were differentiated with unfiltered retinoic acid and cultured in atrial engineered heart tissue. Force and action potential were measured in atrial engineered heart tissues. Single human induced pluripotent stem cell-derived atrial cardiomyocytes were isolated from atrial engineered heart tissue for ion current measurements. RESULTS: PITX2-/- atrial engineered heart tissue beats slightly slower than isogenic control without irregularity. Force was lower in PITX2-/- than in isogenic control (0.053±0.015 versus 0.131±0.017 mN, n=28/3 versus n=28/4, PITX2-/- versus isogenic control; P<0.0001), accompanied by lower expression of CACNA1C and lower L-type Ca2+ current density. Early repolarization was weaker (action potential duration at 20% repolarization; 45.5±13.2 versus 8.6±5.3 ms, n=18/3 versus n=12/4, PITX2-/- versus isogenic control; P<0.0001), and maximum diastolic potential was more negative (-78.3±3.1 versus -69.7±0.6 mV, n=18/3 versus n=12/4, PITX2-/- versus isogenic control; P=0.001), despite normal inward rectifier currents (both IK1 and IK,ACh) and carbachol-induced shortening of action potential duration. CONCLUSIONS: Complete PITX2 deficiency in human induced pluripotent stem cell-derived atrial cardiomyocytes recapitulates some findings of electrical remodeling of atrial fibrillation in the absence of fast beating, indicating that these abnormalities could be primary consequences of lower PITX2 levels.

Resilient Hippocampal Gamma Rhythmogenesis and Parvalbumin-Expressing Interneuron Function Before and After Plaque Burden in 5xFAD Alzheimer's Disease Model.

Recent studies have implicated impaired Parvalbumin Fast-Spiking Interneuron (PVIN) function as a precipitating factor underlying abnormalities in network synchrony, oscillatory rhythms, and cognition associated with Alzheimer's disease (AD). However, a complete developmental investigation of potential gamma deficits, induced by commonly used carbachol or kainate in ex vivo slice preparations, within AD model mice is lacking. We examined gamma oscillations using field recordings in acute hippocampal slices from 5xFAD and control mice, through the period of developing pathology, starting at 3 months of age, when there is minimal plaque presence in the hippocampus, through to 12+ months of age, when plaque burden is high. In addition, we examined PVIN participation in gamma rhythms using targeted cell-attached recordings of genetically-reported PVINs, in both wild type and mutant mice. In parallel, a developmental immunohistochemical characterisation probing the PVIN-associated expression of PV and perineuronal nets (PNNs) was compared between control and 5xFAD mice. Remarkably, this comprehensive longitudinal evaluation failed to reveal any obvious correlations between PVIN deficits (electrical and molecular), circuit rhythmogenesis (gamma frequency and power), and Aß deposits/plaque formation. By 6-12 months, 5xFAD animals have extensive plaque formation throughout the hippocampus. However, a deficit in gamma oscillatory power was only evident in the oldest 5xFAD animals (12+ months), and only when using kainate, and not carbachol, to induce the oscillations. We found no difference in PV firing or phase preference during kainate-induced oscillations in younger or older 5xFAD mice compared to control, and a reduction of PV and PNNs only in the oldest 5xFAD mice. The lack of a clear relationship between PVIN function, network rhythmicity, and plaque formation in our study highlights an unexpected resilience in PVIN function in the face of extensive plaque pathology associated with this model, calling into question the presumptive link between PVIN pathology and Alzheimer's progression.

Diurnal rhythm regulates the frequency of carbachol-induced beta oscillation via inhibitory neural system in rat hippocampus.

Animals have a diurnal rhythm with a cycle of approximately 1 day modulated by light information. This rhythm modulates memory performance. Relatedly, the hippocampal neural circuit has a dynamic property that can induce theta, beta, and gamma brain waves. However, the associated between the diurnal rhythm and these waves has not yet been elucidated. Carbachol, a cholinergic agent, can induce oscillations (e.g., beta waves) in rat hippocampal slices. In this study, we investigate the diurnal changes in the dynamic properties of hippocampal neuronal circuits using carbachol-induced beta oscillations (CIBOs). The hippocampal slices were made from rats adapted to 12-h-each light and dark conditions. The frequency of CIBO was significantly decreased more at midnight than at midday. There was no significant difference both under 12-h-each dark/dark condition and in the shuffled data of diurnal condition. The frequency at midday was significantly decreased by the application of SR95531 (gabazine) and bicuculline gamma-aminobutyric acid (GABA)-A receptor antagonists. In paired-pulse inhibition (PPI) experiments, the PPI ratio at midnight was larger than that at midday. The PPI ratio reflects the degree of recurrent inhibition. The expression level of Glutamate decarboxylase 65, an enzyme that synthetizes GABA, was significantly higher at midnight than at midday. These results suggest that the CIBO frequency can be modulated by diurnal changes of hippocampal inhibitory neurons, and the modulation may lead to a diurnal change in memory performance. Supplementary Information: The online version contains supplementary material available at 10.1007/s11571-021-09736-4.

A vitamin E long-chain metabolite and the inspired drug candidate α-amplexichromanol relieve asthma features in an experimental model of allergen sensitization.

Benefits for vitamin E intake in diseases with inflammatory components have been described and related in part, to endogenously formed metabolites (long-chain metabolites, LCM). Here, we have evaluated the role of LCM in relieving asthma features. To this aim, the endogenous vitamin E metabolite α-13'-carboxychromanol (α-T-13'-COOH) that acts as potent 5-lipoxygenase inhibitor has been administered either intraperitoneally or by oral gavage to BALB/c mice sensitized by subcutaneous injection of ovalbumin (OVA). We also have taken advantage of the metabolically stable α-T-13'-COOH derivative α-amplexichromanol (α-AC). Intraperitoneal treatment with α-T-13'-COOH reduced OVA-induced airway hyperreactivity (AHR) as well as peri-bronchial inflammatory cell infiltration. α-AC was more efficacious than α-T-13'-COOH, as demonstrated by better control of AHR and in reducing subepithelial. Both compounds exerted their protective function by reducing pulmonary leukotriene C4 levels. Beneficial effects of α-AC were coupled to inhibition of the sensitization process, as indicated by a reduction of IgE plasma levels, lung mast cell infiltration and Th2 immune response. Metabololipidomics analysis revealed that α-AC raises the pulmonary levels of prostanoids, their degradation products, and 12/15-lipoxygenase metabolites. Following oral administration, the pharmacodynamically different profile in α-T-13'-COOH and α-AC was abrogated as demonstrated by a similar and improved efficacy in controlling asthma features as well as by metabololipidomics analysis. In conclusion, this study highlights a role for LCM and of vitamin E derivatives as pharmacologically active compounds that ameliorate asthmatic features and defines an important role for endogenous vitamin E metabolites in regulating immune response underlying the sensitization process.

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.

The effectiveness of parasympathomimetics for treating underactive bladder: A systematic review and meta-analysis.

AIMS: Biological rationale suggests that parasympathomimetics (cholinergic receptor stimulating agents) could be beneficial for patients with underactive bladder. However, no systematic review with meta-analysis addressing potential benefits or adverse effects exists. The aim of this review was to assess the effectiveness, both benefits and harms, of using parasympathomimetics for the treatment of underactive bladder. METHODS: The protocol was registered in PROSPERO, and searches undertaken in PubMed, Embase, and CENTRAL, including randomized and non-randomized controlled trials of patients with underactive bladder, comparing parasympathomimetic to placebo, no treatment, or other pharmaceuticals. Risk ratios, odds ratios, and mean differences were calculated. RESULTS: Twelve trials with 3024 participants were included. There was a significant difference between parasympathomimetics and comparators (favoring parasympathomimetics) in the number of patients with urinary retention (risk ratio 0.55, 95% confidence interval [CI] 0.3-0.98, p = 0.04, low quality of evidence). There was no difference in mean postvoid volume overall (MD -41.4 ml, 95% CI -92.0 to 9.1, p = 0.11, low quality of evidence). There was a significant difference at up to 1 week post-intervention, favoring parasympathomimetics (MD -77.5 ml, 95% CI -90.9 to -64.1, p < 0.001, low quality of evidence), but no difference at 1 month post-intervention. There was no difference in adverse events (odds ratio 1.19, 95% CI 0.62-2.28, p = 0.6, moderate quality of evidence). CONCLUSIONS: The evidence supporting the use of parasympathomimetics is of low quality, with relatively short follow-up durations. Overall, it is not possible to draw clear evidence-based conclusions from the current literature, presenting the use of parasympathomimetics for treating underactive bladder as a key area that requires future well-controlled clinical trials.

Water Drinking Behavior Associated with Aversive Arousal in Rats: An Integrative Approach.

Cholinergic muscarinic stimulation of vast areas of the limbic brain induced a well-documented polydipsia in laboratory rats. This excessive water-drinking behavior has not received any convincing biological and physiological interpretation for the last 50 years. This review offers such an interpretation and suggests that cholinergically induced drinking response, mostly by carbachol, is associated with activation of the ascending mesolimbic cholinergic system that serves for initiation of emotional aversive arousal of the organism. The ascending cholinergic system originates from the laterodorsal tegmental nucleus, has a diffuse nature, and affects numerous subcortical limbic structures. It is proposed that the carbachol-induced drinking response is related to the state of anxiety and does not serve the regulation of thirst. Instead, the response is anxiety-induced polydipsia that might occur as a soothing procedure that decreases the aversiveness of the negative emotional state induced by carbachol. It is concluded that carbachol-induced water-drinking behavior is a rewarding process that contributes to alleviating the feeling of anxiety by bringing some relief from the cholinergically induced aversive state, and it is a homologue to anxiety-driven polydipsia in humans.

Effects of Microbeam Irradiation on Rodent Esophageal Smooth Muscle Contraction.

BACKGROUND: High-dose-rate radiotherapy has shown promising results with respect to normal tissue preservation. We developed an ex vivo model to study the physiological effects of experimental radiotherapy in the rodent esophageal smooth muscle. METHODS: We assessed the physiological parameters of the esophageal function in ex vivo preparations of the proximal, middle, and distal segments in the organ bath. High-dose-rate synchrotron irradiation was conducted using both the microbeam irradiation (MBI) technique with peak doses greater than 200 Gy and broadbeam irradiation (BBI) with doses ranging between 3.5-4 Gy. RESULTS: Neither MBI nor BBI affected the function of the contractile apparatus. While peak latency and maximal force change were not affected in the BBI group, and no changes were seen in the proximal esophagus segments after MBI, a significant increase in peak latency and a decrease in maximal force change was observed in the middle and distal esophageal segments. CONCLUSION: No severe changes in physiological parameters of esophageal contraction were determined after high-dose-rate radiotherapy in our model, but our results indicate a delayed esophageal function. From the clinical perspective, the observed increase in peak latency and decreased maximal force change may indicate delayed esophageal transit.

Phytochemical Analysis, Antispasmodic, Myorelaxant, and Antioxidant Effect of Dysphania ambrosioides (L.) Mosyakin and Clemants Flower Hydroethanolic Extracts and Its Chloroform and Ethyl Acetate Fractions.

Dysphania ambrosioides (L.) Mosyakin and Clemants is an annual or ephemeral perennial herb used traditionally in the Mediterranean region in folk medicine to treat various illnesses, including those related to the digestive system. This study aims to assess the antispasmodic, myorelaxant, and antioxidant effects of D. ambrosioides flower hydroethanolic extract and its chloroform and ethyl acetate fractions in a comparative study to evaluate the result of the extraction type on the potential activity of the extract. Both rat and rabbit jejunum were used to evaluate the antispasmodic and myorelaxant effect, while the antioxidant effect was evaluated using DPPH, a ferric reducing power assay, and a beta-carotene bleaching test. LC/MS-MS analysis was carried out to reveal the composition of the different types of extract. Following the results, the hydroethanolic extract showed a significant myorelaxant effect (IC50 = 0.39 ± 0.01 mg/mL). Moreover, it was shown that the hydroethanolic extract demonstrated the best antispasmodic activity (IC50 = 0.51 ± 0.05 mg/mL), followed by the ethyl acetate (IC50 = 4.05 ± 0.32 mg/mL) and chloroform (IC50 = 4.34 ± 0.45 mg/mL) fractions. The antioxidant tests showed that the hydroethanolic extract demonstrated high antioxidant activity, followed by the ethyl acetate and chloroform fractions. The LC/MS-MS analysis indicates that the plant extract was rich in flavonoids, to which the extract activity has been attributed. This study supports the traditional use of this plant to treat digestive problems, especially those with spasms.

Inhibition of Matrix Metalloproteinase 9 Activity Promotes Synaptogenesis in the Hippocampus.

Information coding in the hippocampus relies on the interplay between various neuronal ensembles. We discovered that the application of a cholinergic agonist, carbachol (Cch), which triggers oscillatory activity in the gamma range, induces the activity of matrix metalloproteinase 9 (MMP-9)-an enzyme necessary for the maintenance of synaptic plasticity. Using electrophysiological recordings in hippocampal organotypic slices, we show that Cch potentiates the frequency of miniature inhibitory and excitatory postsynaptic currents (mIPSCs and mEPSCs, respectively) in CA1 neurons and this effect is MMP-9 dependent. Interestingly, though MMP-9 inhibition prevents the potentiation of inhibitory events, it further boosts the frequency of excitatory mEPSCs. Such enhancement of the frequency of excitatory events is a result of increased synaptogenesis onto CA1 neurons. Thus, the function of MMP-9 in cholinergically induced plasticity in the hippocampus is to maintain the fine-tuned balance between the excitatory and the inhibitory synaptic transmission.

Disruption of circadian timing increases synaptic inhibition and reduces cholinergic responsiveness in the dentate gyrus.

We investigated synaptic mechanisms in the hippocampus that could explain how loss of circadian timing leads to impairments in spatial and recognition memory. Experiments were performed in hippocampal slices from Siberian hamsters (Phodopus sungorus) because, unlike mice and rats, their circadian rhythms are easily eliminated without modifications to their genome and without surgical manipulations, thereby leaving neuronal circuits intact. Recordings of excitatory postsynaptic field potentials and population spikes in area CA1 and dentate gyrus granule cells revealed no effect of circadian arrhythmia on basic functions of synaptic circuitry, including long-term potentiation. However, dentate granule cells from circadian-arrhythmic animals maintained a more depolarized resting membrane potential than cells from circadian-intact animals; a significantly greater proportion of these cells depolarized in response to the cholinergic agonist carbachol (10 µM), and did so by increasing their membrane potential three-fold greater than cells from the control (entrained) group. Dentate granule cells from arrhythmic animals also exhibited higher levels of tonic inhibition, as measured by the frequency of spontaneous inhibitory postsynaptic potentials. Carbachol also decreased stimulus-evoked synaptic excitation in dentate granule cells from both intact and arrhythmic animals as expected, but reduced stimulus-evoked synaptic inhibition only in cells from control hamsters. These findings show that loss of circadian timing is accompanied by greater tonic inhibition, and increased synaptic inhibition in response to muscarinic receptor activation in dentate granule cells. Increased inhibition would likely attenuate excitation in dentate-CA3 microcircuits, which in turn might explain the spatial memory deficits previously observed in circadian-arrhythmic hamsters.

BRET- and fluorescence anisotropy-based assays for real-time monitoring of ligand binding to M2 muscarinic acetylcholine receptors.

BRET and fluorescence anisotropy (FA) are two fluorescence-based techniques used for the characterization of ligand binding to G protein-coupled receptors (GPCRs) and both allow monitoring of ligand binding in real time. In this study, we present the first direct comparison of BRET-based and FA-based binding assays using the human M2 muscarinic acetylcholine receptor (M2R) and two TAMRA (5-carboxytetramethylrhodamine)-labeled fluorescent ligands as a model system. The determined fluorescent ligand affinities from both assays were in good agreement with results obtained from radioligand competition binding experiments. The assays yielded real-time kinetic binding data revealing differences in the mechanism of binding for the investigated fluorescent probes. Furthermore, the investigation of various unlabeled M2R ligands yielded pharmacological profiles in accordance with earlier reported data. Taken together, this study showed that BRET- and FA-based binding assays represent valuable alternatives to radioactivity-based methods for screening purposes and for a precise characterization of binding kinetics supporting the exploration of binding mechanisms.

Cannabidiol selectively inhibits the contraction of rat small resistance arteries: Possible role for CGRP and voltage-gated calcium channels.

The pharmacology of cannabidiol, the non-psychoactive major component of Cannabis sativa, is of growing interest as it becomes more widely prescribed. This study aimed to examine the effects of cannabidiol on a wide range of contractile agents in rat small resistance arteries, in comparison with large arteries, and to explore its mechanism of action. The vascular actions of cannabidiol were also contrasted with effects on the contractions of bronchial, urogenital, cardiac and skeletal muscles. Isolated small or large arteries were incubated with cannabidiol (0.3-3 µM) or vehicle and concentration-contraction response curves were completed to various agents, including endothelin-1, arginine vasopressin, methoxamine, 5-HT, α-methyl 5-HT and U46619. In small arteries, the effects of cannabidiol were tested in the presence of antagonists of CB1 or CB2 receptors, calcitonin gene-related peptide (CGRP), nitric oxide synthase, cyclooxygenase, PPARγ or a combination. The role of L-type voltage-operated calcium channels was also assessed. Cannabidiol 1-3 µM significantly inhibited the contraction of small resistance arteries to all tested agents through a combination of mechanisms that include CGRP and L-type calcium channels. However, large arteries were insensitive to cannabidiol. Cannabidiol (10-100 µM) was largely without effect in bronchi, atria and hemidiaphragm, but 100 µM attenuated maximum contractions in vasa deferentia. Cannabidiol's effects in the clinical range (1-3 µM) appear to be specific to small resistance arteries. This high sensitivity of the resistance arterial circulation to cannabidiol may offer a therapeutic opportunity in peripheral vascular disease that excludes off-target sites such as the heart and non-vascular smooth muscle.

The pretreatment of rats with nebivolol ameliorates bladder contractile dysfunction caused by ischemia-reperfusion injury.

OBJECTIVE: The present study aimed to investigate the protective effect of nebivolol in the bladder isolated from rats exposed to ischemia-reperfusion (IR) injury. METHODS: Sprague-Dawley rats were divided into control, IR, and nebivolol+IR groups. In the nebivolol+IR group, nebivolol was administered (0.4 mg/kg, subcutaneous) in rats prior to IR insult. At the end of the experimental protocol, the urinary bladder was rapidly isolated and bladder strips were mounted in an organ bath. After the equilibration period, potassium chloride (KCl, 20-100 mM) or carbachol (0.01-10 µM) was cumulatively added to the organ bath to generate cumulative concentration-response curves (CCRCs). Oxidative stress and interleukin 6 (IL-6) levels were also evaluated in the bladder tissue. RESULTS: The CCRCs of KCl and carbachol were significantly reduced in the IR group compared to those of the control, and this inhibition was reversed by the pretreatment of rats with nebivolol (P < .05). The IR group's total antioxidant status was significantly lower with a concomitant increase in IL-6 levels than that of the control and nebivolol+IR groups (P < .05). CONCLUSIONS: The present study indicates that pretreatment of rats with nebivolol (0.4 mg/kg) could improve bladder contractile dysfunction caused by IR injury through suppression of increased oxidative stress and IL-6 levels.

KV7 Channel Expression and Function Within Rat Mesenteric Endothelial Cells.

Background and Purpose: Arterial diameter is dictated by the contractile state of the vascular smooth muscle cells (VSMCs), which is modulated by direct and indirect inputs from endothelial cells (ECs). Modulators of KCNQ-encoded kV7 channels have considerable impact on arterial diameter and these channels are known to be expressed in VSMCs but not yet defined in ECs. However, expression of kV7 channels in ECs would add an extra level of vascular control. This study aims to characterize the expression and function of KV7 channels within rat mesenteric artery ECs. Experimental Approach: In rat mesenteric artery, KCNQ transcript and KV7 channel protein expression were determined via RT-qPCR, immunocytochemistry, immunohistochemistry and immunoelectron microscopy. Wire myography was used to determine vascular reactivity. Key Results: KCNQ transcript was identified in isolated ECs and VSMCs. KV7.1, KV7.4 and KV7.5 protein expression was determined in both isolated EC and VSMC and in whole vessels. Removal of ECs attenuated vasorelaxation to two structurally different KV7.2-5 activators S-1 and ML213. KIR2 blockers ML133, and BaCl2 also attenuated S-1 or ML213-mediated vasorelaxation in an endothelium-dependent process. KV7 inhibition attenuated receptor-dependent nitric oxide (NO)-mediated vasorelaxation to carbachol, but had no impact on relaxation to the NO donor, SNP. Conclusion and Implications: In rat mesenteric artery ECs, KV7.4 and KV7.5 channels are expressed, functionally interact with endothelial KIR2.x channels and contribute to endogenous eNOS-mediated relaxation. This study identifies KV7 channels as novel functional channels within rat mesenteric ECs and suggests that these channels are involved in NO release from the endothelium of these vessels.

Carbachol and Nicotine in Prefrontal Cortex Have Differential Effects on Sleep-Wake States.

The role of the brainstem cholinergic system in the regulation of sleep-wake states has been studied extensively but relatively little is known about the role of cholinergic mechanisms in prefrontal cortex in the regulation of sleep-wake states. In a recent study, we showed that prefrontal cholinergic stimulation in anesthetized rat can reverse the traits associated with anesthesia and restore a wake-like state, thereby providing evidence for a causal role for prefrontal cholinergic mechanisms in modulating level of arousal. However, the effect of increase in prefrontal cholinergic tone on spontaneous sleep-wake states has yet to be demonstrated. Therefore, in this study, we tested the hypothesis that delivery of cholinergic agonists - carbachol or nicotine - into prefrontal cortex of rat during slow wave sleep (SWS) would produce behavioral arousal and increase the time spent in wake state. We show that unilateral microinjection (200 nL) of carbachol (1 mM) or nicotine (100 mM) into prefrontal cortex during SWS decreased the latency to the onset of wake state (p = 0.03 for carbachol, p = 0.03 for nicotine) and increased the latency to the onset of rapid eye movement sleep (p = 0.008 for carbachol, p = 0.006 for nicotine). Although the infusion of 1 mM carbachol increased the time spent in wake state (p = 0.01) and decreased the time spent in SWS (p = 0.01), infusion of 10 or 100 mM nicotine did not produce any statistically significant change in sleep-wake architecture. These data demonstrate a differential role of prefrontal cholinergic receptors in modulating spontaneous sleep-wake states.