Carbachol-induced Contraction Research Articles

Secretin Inhibits Small Intestinal Motility via Interstitial Cells of Cajal and cAMP Mechanisms

Background: Secretin is a member of the secretin-glucagon-vasoactive intestinal peptide hormone superfamily and is a multifunctional gastrointestinal- (GI) and neuro- peptide hormone. Secretin is primarily secreted postprandially from the crypts of Lieberkühn of duodenal enteroendocrine S cells into circulation where it reaches secretin receptor targets in the central nervous system and periphery. This dynamic hormone has been shown to act as a key signaling molecule in the regulation of digestion, metabolism and energy expenditure, water retention, reproduction, thermogenesis in adipose tissue, and in gastric and intestinal motility. Secretin’s canonical role in the GI tract is to stimulate the secretion of bicarbonate and bile from pancreatic ducts and bile ducts to neutralize acidic chyme exiting the stomach. Secretin has also been shown to slow intestinal motility, but its targets and mechanism of action is poorly understood. Aims: Several studies have proposed that secretin acts to slow intestinal motility in the intestines primarily through the many secretin receptors present on vagal afferents in the GI tract, here we discuss new data that suggests an alternate and complementary signaling pathway via interstitial cells of Cajal (ICC). ICC act as a liaison to facilitate a reduction in force of GI smooth muscle contraction through the activation of the secretin receptor (SCTR) and subsequent stimulation of the second messenger, cyclic adenosine monophosphate (cAMP). Here we provide evidence to show how ICC mediates changes in myogenic activity and motility in the small intestine. Methods: Spinning-disk confocal microscopy was used to monitor Ca2+ signaling in ICC from small intestinal muscles of GCaMP6f x KitiCre mice. Additionally, cAMP levels were evaluated using CAMPER mice. Intestinal muscle contractility was assessed using muscle strip myography experiments. Results: Secretin reduced small intestinal force of contraction in the presence of tetrodotoxin (TTX) and dampened the effect of cholinergic transmission. The secretin receptor (SCTR) is expressed primarily on ICC, specifically ICC within the deep muscular plexus (ICC-DMP) in the small intestine and Ca2+ imaging confirmed the effects are primarily localized within ICC-DMP. Secretin reduced carbachol-induced contractions and Ca2+ transients in ICC-DMP in response to electrical field stimulation (EFS) in the presence of LNNA (NO synthase inhibitor) and MRS2500 (P2Y1 antagonist). Secretin caused an increase in cAMP levels in ICC-DMP in muscles from Kit-iCre-CAMPER mice. PKA inhibitors rescued some of the effects of secretin on ICC-DMP Ca2+ signaling. Measurements of diameter change in large, intact, intestinal segments (4-5 cm) showed a significant decrease in response to Secretin. Conclusions: Secretin can inhibit small intestinal motility through the activation SCTRs on ICC-DMP via cAMP-mediated mechanisms. These results show how novel secretin targets on ICC influence GI muscles and reduce propulsive and segmental motility to facilitate nutrient absorption and digestion after a meal. Funding: This project was supported by R01 DK-120759 from the National Institute of Diabetes and digestive and Kidney (NIDDK). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Relaxant Activity of 4H-Pyran and 1,6-Dihydropyridine Derivatives on Isolated Rat Trachea

Derivatives of 4H-pyrans and 1,6-dihydropyridines have generated considerable attention due to their interesting biological and therapeutic values. Their pharmacological activities include vasorelaxant, anticarcinogenic, antimicrobial, and antioxidant activities. Thus, the aim of the current work is to determine the relaxant effect of synthesized 4H-pyran and 1,6-dihydropyridine derivatives with potential anti-asthmatic properties on the smooth muscle airway, with a possible Ca2+-channel blockade as a mechanism of action due to their analogy with 1,4-dihidropyridines. 4H-pyrans and 1,6-dihydropyridines were achieved using multicomponent reactions by microwave and conventional heating. Also, test samples were evaluated ex vivo to determine their relaxant effect on isolated rat tracheal rings pre-contracted with carbachol. All compounds evaluated showed a significant relaxant effect on carbachol-induced contraction in tracheal rat rings. Compounds 4b, 4e, 7a, and 8d were the most potent from the entire series and were also more potent than theophylline, used as a positive control. In conclusion, in the current work some relaxant compounds of the airway smooth muscle with potential to be developed as anti-asthmatic drugs were obtained.

Spasmolytic Effect of Flopropione Does Not Involve Catechol-O- methyltransferase (COMT) Inhibition.

Flopropione (Flo) has been used for gallstone and urolithiasis as a spasmolytic agent almost exclusively in Japan. According to the package insert, its main mechanism is catechol-O-methyltransferase (COMT) inhibition and anti-serotonergic effect. This is obviously contrary to pharmacological common sense, but it is described that way in pharmacology textbooks and occurs in questions in the National Examination for Pharmacists in Japan. As this is a serious problem in education, we re-examined the action of Flo. The guinea pig ureter was hardly contracted by serotonin, but noradrenaline (NA) elicited repetitive twitch contraction, which was inhibited by Flo. The sphincter of Oddi (SO) exhibited a spontaneous repetitive twitch contraction, which was inhibited by NA and Flo. The inhibitory effect of NA was reversed by α- and β-blockers, whereas that of Flo was not. Entacapone, a representative COMT inhibitor, did not affect the movement of the ureter and the SO. Nifedipine suppressed carbachol-induced contraction of the taenia coli, spontaneous movement of the SO, and NA-induced contraction of the ureter to almost the same extent, whereas Flo did not inhibit the taenia coli, but inhibited the contraction of the SO and the ureter. The inhibitory pattern of Flo resembled that of the ryanodine receptor agonist 4-chloro-m-cresol and the inositol 1,4,5-trisphosphate (IP3) receptor antagonist 2-aminoethoxydiphenyl borate. It is concluded that COMT inhibition or serotonin inhibition is not involved in the spasmolytic action of Flo. Flo might act on ryanodine receptors and/or IP3 receptors, which are responsible for periodic Ca release from Ca stores, to disrupt coordinated Ca dynamics.

Hydrogen Sulfide (H<sub>2</sub>S) and Reactive Oxygen Species (ROS) Scavengers Have a Protective Effect on Carbachol-induced Contractions That are Impaired by High Glucose in Detrusor Smooth Muscle

Hydrogen Sulfide (H<sub>2</sub>S) and Reactive Oxygen Species (ROS) Scavengers Have a Protective Effect on Carbachol-induced Contractions That are Impaired by High Glucose in Detrusor Smooth Muscle

Electrophysiology, molecular modelling, and functional analysis of the effects of dietary quercetin and flavonoid analogues on Kir6.1 channels in rat stomach fundus smooth muscle

Flavonoids, ubiquitously distributed in the plant world, are regularly ingested with diets rich in fruit, vegetables, wine, and tea. During digestion, they are partially absorbed in the stomach. The present work aimed to assess the in vitro effects of quercetin and ten structurally related flavonoids on the rat gastric fundus smooth muscle, focussing on ATP-dependent K+ (Kir6.1) channels, which play a central role in the regulation of resting membrane potential, membrane excitability and, consequently, of gastric motility. Whole-cell currents through Kir6.1 channels (IKir6.1) were recorded with the patch-clamp technique and the mechanical activity of gastric fundus smooth muscle strips was studied under isometric conditions. Galangin ≈ tamarixetin > quercetin > kaempferol > isorhamnetin ≈ luteolin ≈ fisetin > (±)-taxifolin inhibited pinacidil-evoked, glibenclamide-sensitive IKir6.1 in a concentration-dependent manner. Morin, rutin, and myricetin were ineffective. The steric hindrance of the molecule and the number and position of hydroxyl groups on the B ring played an important role in the activity of the molecule. Molecular docking simulations revealed a possible binding site for flavonoids in the C-terminal domain of the Kir6.1 channel subunit SUR2B, in a flexible loop formed by residues 251 to 254 of chains C and D. Galangin and tamarixetin, but not rutin relaxed both high K+- and carbachol-induced contraction of fundus strips in a concentration-dependent manner. Furthermore, both flavonoids shifted to the right the concentration-relaxation curves to either pinacidil or L-cysteine constructed in strips pre-contracted by high K+, rutin being ineffective. In conclusion, IKir6.1 inhibition exerted by dietary flavonoids might counterbalance their myorelaxant activity, affect gastric accommodation or, at least, some stages of digestion.

Age-dependent effects of the β3 adrenoceptor agonist CL316,243 on human and rat detrusor muscle strips

Motility of detrusor smooth muscle includes adrenergic relaxation and cholinergic contraction. Since the latter may be deregulated in overactive bladder (OAB) pathophysiology, anticholinergics are the standard therapy but occasionally less tolerated due to side effects such as dry mouth and constipation. β3 adrenoceptor agonists also alleviate OAB symptoms by relaxing the detrusor muscle. Their age dependence, however, is far from understood. To address this issue, we induced contractions with KCl (60 mM) and carbachol (from 10 nM to 100 μM) in the presence of the β3 adrenoceptor agonist CL316,243 (from 0.1 to 10 μM) in both human and rat muscle strips. Our results confirmed that both contractions were attenuated by β3 adrenoceptor activation in both species, but with differing age dependence. In humans, specimens from mid-life subjects showed a significantly more pronounced effect of CL316,243 in attenuating carbachol-induced contractions than those from aged subjects (Cohen’s d of maximal attenuation: 1.82 in mid-life versus 0.13 in aged) without altering EC50. Conversely, attenuation of KCl responses by CL316,243 increased during ageing (Spearman correlation coefficient = -0.584, P<0.01). In rats, both KCl- and carbachol-induced contractions were significantly more attenuated by CL316,243 in samples from adolescent as compared to aged samples. Immunohistochemistry in human detrusor sections proved β3 adrenoreceptor abundance to remain unaltered during ageing. In conclusion, our findings suggest differential age-dependent changes in human β3 adrenoceptor-dependent attenuation of detrusor contraction in terms of electromechanical versus pharmacomechanical coupling; they may help understand the differential responsiveness of OAB patients to β3 agents.

Role of nitric oxide and calcium ions in the effects of hydrogen sulfide on contractile activity of rat jejunum

This study was performed to explore the role of nitric oxide, intracellular and extracellular calcium in the effects of hydrogen sulfide on spontaneous and carbachol-induced contractions of a rat jejunum preparation during a isometric contraction. Application of H2S donor, sodium hydrosulfide, led to a decrease in tonic tension, the amplitude and frequency of spontaneous contractions, as well as in the amplitude induced by carbachol, a nonspecific acetylcholine receptor agonist. Inhibiting the production of endogenous NO synthesis by with L-NAME, the effect of H2S donor remained unchanged, while in the presence of SNAP, a NO donor, the effects of NaHS on the amplitude of spontaneous and carbachol-induced contractions were less pronounced. Dantrolene, a ryanodine receptor inhibitor was used to stop a decrease in tonic tension in the presence of NaHS. The calcium-free solution reduced the inhibitory effect of NaHS on carbachol-induced contractions. This suggests that the inhibitory effect of H2S is associated with the dynamics of the intracellular concentration of calcium ions, and the interaction between NO and H2S occurs at the level of common targets of two gases.

Анестетик лідокаїн пригнічує холінергічні скорочення тонкої кишки миші та спричиняє порушення шлунково-кишкового тракту людини

Lidocaine is one of the components of multimodal low-opioid anesthesia, which is commonly used in surgical procedures, especially in cardiac surgery. Lidocaine has been used in medicine for a long time as a local anesthetic, but after the invention of the method of its intravenous administration, questions arose about its possible side effects on the visceral system, in particular on the motility of the gastrointestinal tract. Therefore, the aim of our work was to investigate the main aspects of the use of lidocaine-based opioid-free anesthesia and its side effects and also to study the effect of lidocaine on the contractile activity of small intestine smooth muscle, namely the mouse ileum. We recorded the contractile activity of smooth muscles of the ileum of mice using the tensiometry method and found that lidocaine at the clinically relevant concentration range, i.e. 1.5, 3, and 5 μg/ml, inhibited carbachol-induced contractions by 16, 27 and 37%, respectively (n = 7). To determine the side effects of opioid-free anesthesia, we studied 60 patients with coronary artery disease undergoing coronary artery stenting with the administration of anesthesia based on lidocaine solution at different concentrations (1 and 2 mg/kg). The side effects included nausea and vomiting, which may indicate а disturbance of gastrointestinal motility, as well as numbness of the limbs, cheeks, tongue, etc. These results contribute to a better understanding of the spectrum of action of lidocaine, a popular anesthetic in surgery, on the visceral system and become an important basis for the future development of new schemes for the use of local anesthetics in clinical practice, particularly in surgical procedures.

Organ-specific off-target effects of Pim/ZIP kinase inhibitors suggest lack of contractile Pim kinase activity in prostate, bladder, and vascular smooth muscle.

Smooth muscle contraction by Pim kinases and ZIPK has been suggested, but evidence for lower urinary tract organs or using Pim-selective inhibitor concentrations is not yet available. Here, we assessed effects of the Pim inhibitors AZD1208 and TCS PIM-1 and the dual ZIPK/Pim inhibitor HS38 on contractions of human prostate and bladder tissues and of porcine interlobar arteries. Human tissues were obtained from radical prostatectomy and radical cystectomy and renal interlobar arteries from pigs. Contractions were studied in an organ bath. Noradrenaline-, phenylephrine- and methoxamine-induced contractions were reduced (up to > 50%) with 500-nM AZD1208 in prostate tissues and to lesser degree and not consistently with all agonists in interlobar arteries. A total of 100-nM AZD1208 or 500-nM TCS PIM-1 did not affect agonist-induced contractions in prostate tissues. Decreases in agonist-induced contractions with 3-µM HS38 in prostate tissues and interlobar arteries were of small extent and did not occur with each agonist. Carbachol-induced contractions in detrusor tissues were unchanged with AZD1208 (500nM) or HS38. Electric field stimulation-induced contractions were not affected with AZD1208 or HS38 in any tissue, but slightly reduced with 500-nM TCS PIM-1 in prostate tissues. Concentration-dependent effects of Pim inhibitors suggest lacking Pim-driven smooth muscle contraction in the prostate, bladder, and interlobar arteries but point to organ-specific functions of off-targets. Procontractile functions of ZIPK in the prostate and interlobar arteries may be limited and are lacking in the detrusor.

Ex Vivo and In Silico Approaches of Tracheal Relaxation through Calcium Channel Blockade of 6-Aminoflavone and Its Toxicological Studies in Murine Models.

Asthma is a condition in which a person's airways become inflamed, narrowed, and produce greater amounts of mucus than normal. It can cause shortness of breath, chest pain, coughing, or wheezing. In some cases, symptoms may be exacerbated. Thus, the current study was designed to determine the mechanism of action of 6-aminoflavone (6-NH2F) in ex vivo experiments, as well as to determine its toxicity in acute and sub-chronic murine models. Tissues were pre-incubated with 6-NH2F, and concentration-response curves to carbachol-induced contraction were constructed. Therefore, tracheal rings pre-treated with glibenclamide, 2-aminopyridine, or isoproterenol were contracted with carbachol (1 µM), then 6-NH2F relaxation curves were obtained. In other sets of experiments, to explore the calcium channel role in the 6-NH2F relaxant action, tissues were contracted with KCl (80 mM), and 6-NH2F was cumulatively added to induce relaxation. On the other hand, tissues were pre-incubated with the test sample, and after that, CaCl2 concentration-response curves were developed. In this context, 6-NH2F induced significant relaxation in ex vivo assays, and the effect showed a non-competitive antagonism pattern. In addition, 6-NH2F significantly relaxed the contraction induced by KCl and CaCl2, suggesting a potential calcium channel blockade, which was corroborated by in silico molecular docking that was used to approximate the mode of interaction with the L-type Ca2+ channel, where 6-NH2F showed lower affinity energy when compared with nifedipine. Finally, toxicological studies revealed that 6-NH2F possesses pharmacological safety, since it did not produce any toxic effect in both acute and sub-acute murine models. In conclusion, 6-aminoflavone exerted significant relaxation through calcium channel blockade, and the compound seems to be safe.

Essential oil of Sicilian Prangos ferulacea (L.) Lindl. and its major component, β−ocimen, affect contractility in rat small and large intestine

Ethnopharmacological relevancePrangos ferulacea (L.) Lindl is an Apiaceae plant, widely used in traditional medicine. Recently, chemical composition and biological activities of its essential oil (Prangroil) have been reported, but there are no studies on possible effects on intestinal contractility. Aims of the studyWe investigated the effects of essential oil Sicilian Prangoil on the contractility of rat small (duodenum) and large (colon) intestine and the related action mechanism. Materials and methodsResponses to Prangoil and to its major component β−ocimen in intestinal segments were assessed in vitro as changes in isometric tension. ResultsPrangoil, induced in duodenum, depending upon doses, contraction and/or muscular relaxation. Instead, in colon Prangoil only reduced the phasic contractions and induced muscular relaxation. β−ocimen, in both segments, produced only reduction of the spontaneous contractions without affecting basal tone. Prangoil contractile effects were abolished by ω-conotoxin, neural N-type Ca2+ channels blocker, atropine, muscarinic receptor antagonist, neostigmine, acetylcholinesterase (AChE) inhibitor, suggesting that Prangoil-induced contraction would be the result of an increase in neuronal cholinergic activity. Prangoil and β−ocimen inhibitory effects were unaffected by ω−conotoxin, L-NAME, blocker of the NO synthase, ODQ, soluble guanylate cyclase inhibitor, excluding involvement of neurotransmitter release or NO synthesis in the inhibitory effects. Potassium channel blocker did not affect Prangoil or β-ocimen inhibitory responses. Prangoil or β-ocimen inhibited the Ca2+ and high-KCl solution -induced contractions and the Carbachol-induced contractions in calcium free solution. ConclusionPrangoil affects the contractility of small and large intestine in rat, with regional differences, via potentiation of neural cholinergic activity, blockade of L-type voltage-gated calcium channel and reduction of Ca2+ release from the intracellular store. The Prangroil main components, β−ocimen, contributes to the inhibitory effects.

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 (10-8-10-4M) induced a concentration-dependent contraction in bladder tissues. L-cysteine (H2S precursor; 10-2M) and exogenous H2S (NaHS; 10-3M) 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 10-2M PAG (~ 40%) and 10-3M AOAA (~ 55%), cystathionine-gamma-lyase (CSE) and cystathionine-β-synthase (CBS) inhibitor, respectively. Y-27632 (10-6M) and GF 109203X (10-6M), 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 ± 0.06nmol/mg respectively), and this augmentation in H2S level decreased with PAG (0.17 ± 0.02, 0.15 ± 0.03, and 0.07 ± 0.04nmol/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.

Dimethyl Sulfoxide Enhances Acetylcholine-Induced Contractions in Rat Urinary Bladder Smooth Muscle by Inhibiting Acetylcholinesterase Activities.

Dimethyl sulfoxide (DMSO) has been used not only as an experimental solvent, but also as a therapeutic agent for interstitial cystitis. The therapeutic effects of DMSO on interstitial cystitis are presumed to involve anti-inflammatory and analgesic effects. However, the effects of DMSO on urinary bladder smooth muscle (UBSM) have not been fully investigated. Thus, in this study, we investigated the effects of DMSO on rat UBSM contractions, and these effects were compared with those of acetone, which has a structure in which the sulfur of DMSO is replaced with carbon. DMSO (0.5-5%) enhanced acetylcholine (ACh)-induced contractions, whereas acetone (3 and 5%) suppressed them. Additionally, DMSO (5%) suppressed carbachol-induced contractions. DMSO/acetone (0.5-5%) inhibited 80 mM KCl-induced contractions in a concentration-dependent manner; however, the inhibitory effects of DMSO were weaker than those of acetone. The enhancing/suppressing effects of DMSO and acetone were almost completely abolished by wash out. DMSO and acetone (0.5-5%) inhibited recombinant human acetylcholinesterase (rhAChE) activity in a concentration-dependent manner. At 0.5 and 1%, the inhibitory effects of DMSO on rhAChE activity were more potent than those of acetone. These findings suggest that DMSO can enhance ACh-induced UBSM contractions and promote urinary bladder motility by inhibiting acetylcholinesterase (AChE), although DMSO also inhibits Ca2+ influx-mediated UBSM contractions. In addition, the sulfur atom in DMSO might play an important role in its enhancing effect on ACh-induced contractions by inhibiting AChE, as acetone did not enhance these contractions.

Effects of ATP on Time Parameters of Contractility of Rats’ Slow and Fast Skeletal Muscles in Normal and Hypothermic Conditions

We have previously shown that hypothermia leads to an increase in the synaptic modulating effects of ATP but not of adenosine in several different animal skeletal muscles. In this paper, we studied the effect of ATP on the amplitude–time parameters of single and tetanic contractions of rats’ isolated fast (1) and slow (2) muscles at different temperatures. We found that when muscles were stimulated by the electrical field (0.1 Hz, 0.5 ms, 10 V), with a decrease in the bath temperature from 37 °C to 14 °C (3), there was an increase in the half-relaxation time of the slow muscle (m. soleus), but not of the fast muscle (m. EDL). Similar effects were observed using a carbachol-induced contraction technique, which suggests the postsynaptic (4) nature of the expansion of the contractile response of the slow muscle induced by ATP (5). To confirm the postsynaptic nature of the observed phenomenon, experiments were performed at a high calcium level (7.2 mM), in which the presynaptic effects of ATP were shown to be offset. We found that the hypercalcium condition did not significantly change the effects of ATP on the measured parameters in both muscles. To record muscle tetanic contractions, we gradually increased the frequency of electrical impulses with the increment of 2.5 Hz to achieve the fusion frequencies of 12.5 Hz for m. soleus and 17.5 Hz for m. EDL at normal temperatures. ATP (100 μM) did not change the fusion frequency for both muscles at a normal temperature but decreased this parameter for the slow muscle to 5 Hz at 14 °C without affecting that for the fast muscle. We conclude that ATP potentiates a hypothermia-induced increase in the half-relaxation time of the contraction of rats’ slow, but not fast, skeletal muscles by acting on postsynaptic P2 receptors (6).

5-HT3 receptors modulate changes in voiding pattern and bladder contractility in water avoidance stress-induced bladder overactivity in male mice.

Chronic psychological stress aggravates painful bladder syndrome symptoms. Previous studies suggest roles of 5-HT3 receptors in regulating micturition and bladder hypersensitivity. This study aimed to investigate the roles of 5-HT3 receptors in modulating voiding patterns and spontaneous bladder contractile properties in water avoidance stress-induced mice. Voiding patterns in sham stress (SS), water avoidance stress (WS), and water avoidance stress with daily oral gavage of ondansetron (1mg/kg BW) (WA) groups were analyzed after exposure to repeated water avoidance stress for 10days. Changes in contractile activity of isolated bladder in response to KCl, carbachol, and 5-hydroxytryptamine were determined. Bladder mast cell quantification was examined using toluidine blue staining. Urine voided area was significantly decreased in WS group after exposure to 10days of the stress protocol, which was reversed in the WA group. The WS group had a higher number of urine spots than the SS group. Increased mast cell degranulation was observed in the stressed mice. Bladder strips of the WS group showed higher tonic and amplitude of spontaneous contraction than the SS group, which were normalized by ondansetron administration. Increased response to carbachol-induced bladder contraction was observed in the bladder of stressed mice, which was attenuated with ondansetron pre-incubation. Water avoidance stress-induced mice exhibited changes in voiding pattern, which was reversed by oral administration with a 5-HT3 receptor antagonist (ondansetron). Enhanced contractile response to cholinergic stimulation in the urinary bladder of the psychological stress-induced bladder overactivity was mediated through 5-HT3 receptors.

Daidzein is the in vivo active compound of Puerariae Lobatae Radix water extract for muscarinic receptor-3 inhibition against overactive bladder.

Background: In the previous study, Puerariae Lobatae Radix (named Gegen in Chinese) water extract attenuated M3 receptor agonist carbachol-induced detrusor contraction after 3-week oral administration in a hypertension-associated OAB (overactive bladder) model. This research aimed to investigate the active ingredients from Gegen water extract against OAB. Methods: Bioassay-guided fractionation was performed by using preparative HPLC for fast isolation of fractions followed by screening their ex vivo activity through carbachol-induced bladder strip contraction assay. Chemicals in each active fraction were analyzed by HPLC-UV. Urine metabolites were quantified by LC-MS/MS after sub-acute administration. Thermal shift assay with the recombinant human M3 receptor protein was performed, and molecular docking analysis was used for molecular modelling of M3 receptor inhibition. Results: Bioassay-guided fractionation results for isolating M3 receptor inhibitors indicated that four compounds were identified as active ingredients of Gegen water extract, and their inhibition potency on carbachol-induced detrusor contraction was ranked in descending order according to their inhibition concentrations as follows: genistein > daidzein > biochanin A >> puerarin. Daidzein in urine reached an ex vivo effective concentration to inhibit detrusor contraction, but others did not. Daidzein concentration-dependently increased the melt temperature (Tm) of recombinant human M3 receptor protein with a positive binding (ΔTm = 2.12 °C at 100 μg/ml). Molecular docking analysis showed that daidzein can potently bind to the ligand binding pocket of the M3 receptor via hydrogen bonding. Conclusion: Puerarin and its derivatives were pro-drugs, and daidzein was their in vivo active form via M3 receptor inhibition for treating OAB.

Aerobic Exercise Reduces Airway Smooth Muscle Contraction In Asthmatic Rats Via Inhibition Of Il-4 Secretion And Suppression Of Store-operated Ca2+ Entry

PURPOSE: Increasing evidence has shown that aerobic exercise training reduces airway hyperresponsiveness in asthmatic individuals. However, the underlying mechanisms remain elusive. This study aimed to investigate the effect of exercise on airway smooth muscle (ASM) contractile function in asthmatic rats and uncover the possible involvement of interleukin 4 (IL-4) and the store-operated Ca2+ entry (SOCE) pathway. METHODS: Chicken ovalbumin was used to stimulate asthma in male Sprague-Dawley rats. The exercise group received moderate-intensity aerobic exercise training for 4 weeks. IL-4 concentrations in bronchoalveolar lavage fluid (BALF) samples were evaluated by ELISA. The contractile function of the ASM was investigated using tracheal ring tension experiments and intracellular Ca2+ imaging techniques. Western-blot experiments were used to identify expression levels of the calcium-release activated calcium (CRAC) channel protein, Orai, in ASM. RESULTS: The carbachol-stimulated and SOCE-mediated contraction of rat ASM was significantly increased in asthmatic rats, but was abolished by exercise training. Pharmacological studies revealed that GSK5498A, a selective blocker of CRAC channels, significantly inhibited SOCE-induced ASM contraction. In addition, our data showed that aerobic exercise inhibits the up-regulation of IL-4 in BALF as well as Orai protein (subtypes 1 and 2) expression in the ASM of asthmatic rats. In line with these observations, we demonstrated that pretreatment of ASM with IL-4 both up-regulated the expression level of Orai (subtype 1, 2, 3) and promoted carbachol-induced and SOCE-mediated ASM contraction. CONCLUSIONS: The present study revealed that aerobic exercise improved ASM contractile function in asthmatic rats by inhibiting IL-4 secretion and down-regulating the expression levels of Orai, thus decreasing excessive SOCE-mediated ASM contraction in asthmatic rats. Supported by Natural Science Foundation of Guangdong Province (2019A1515012167).

Determination of Region-Specific Roles of the M3 Muscarinic Acetylcholine Receptor in Gastrointestinal Motility.

The specific role of the M3 muscarinic acetylcholine receptor in gastrointestinal motility under physiological conditions is unclear, due to a lack of subtype-selective compounds. The objective of this study was to determine the region-specific role of the M3 receptor in gastrointestinal motility. We developed a novel positive allosteric modulator (PAM) for the M3 receptor, PAM-369. The effects of PAM-369 on the carbachol-induced contractile response of porcine esophageal smooth muscle and mouse colonic smooth muscle (ex vivo) and on the transit in mouse small intestine and rat colon (in vivo) were examined. PAM-369 selectively potentiated the M3 receptor under the stimulation of its orthosteric ligands without agonistic or antagonistic activity. Half-maximal effective concentrations of PAM activity for human, mouse, and rat M3 receptors were 0.253, 0.345, and 0.127μM, respectively. PAM-369 enhanced carbachol-induced contraction in porcine esophageal smooth muscle and mouse colonic smooth muscle without causing any contractile responses by itself. The oral administration of 30mg/kg PAM-369 increased the small intestinal transit in both normal motility and loperamide-induced intestinal dysmotility mice but had no effects on the colonic transit, although the M3 receptor mRNA expression is higher in the colon than in the small intestine. This study provided the first direct evidence that the M3 receptor has different region-specific roles in the motility function between the small intestine and colon in physiological and pathophysiological contexts. Selective PAMs designed for targeted subtypes of muscarinic receptors are useful for elucidating the subtype-specific function.

Haloperidol, Olanzapin, Risperidon ve Klozapinin Organ Banyosu Sistemi Kullanılarak Fare Detrusor Kası Üzerine Etkileri

Objective: The bladder normally shows no contractility or activity during the filling phase. In the overactive urinary bladder, spontaneous contractions and detrusor instability are seen in the filling phase and urinary incontinence occurs. This study aims to demonstrate the effects of first-generation antipsychotic haloperidol and second-generation antipsychotics olanzapine, risperidone, and clozapine on mice isolated bladder using the organ bath system. Materials and Methods: 63 male inbred mice were divided as saline, haloperidol 0.125 mg/kg, haloperidol 0.25 mg/kg, olanzapine 1 mg/kg, olanzapine 2 mg/kg, risperidone 0.25 mg/kg, risperidone 0.5 mg/kg, clozapine 1.25 mg/kg and clozapine 2.5 mg/kg groups. Mice were treated with drugs for 21 days. Then, the effects of drugs were investigated on isoproterenol-induced relaxation responses of carbachol-induced contractions in isolated detrusor strips. Results: We showed that carbachol-induced contractions relaxed by isoproterenol and papaverine in mice detrusor strips obtained from olanzapine, risperidone, and clozapine treated groups. There were no significant differences in KCl-induced contractile responses among the groups.Conclusion: Olanzapine, risperidone, and clozapine increased the isoproterenol-induced relaxations of the detrusor muscle that increased the bladder capacity. These drugs might be clinically useful for the treatment of overactive urinary bladder in patients that should use antipsychotic drugs.

Therapeutic potential of Ceratonia siliqua extract for the management of asthma and hypertension.

The current work was performed to explore the pharmacological mechanisms involved in the management of asthma and hypertension along with the safety profile of the Ceratonia siliqua (C. siliqua/Carob) pods. The bronchorelaxant, vasorelaxant, and cardioselective activities of C. siliqua pods were investigated using isolated rabbit tracheal, aortic, and paired atrial fragments on the Power lab data acquisition system. Normotensive rats were used to study antihypertensive activity. The plant extract and its fractions relaxed the carbachol-induced contraction in the tracheal fragments and shifted the concentration-response curve of carbachol towards the right confirming the muscarinic receptor antagonist activity. The relaxation of phenylephrine-induced contraction in an aortic fragment by the extract showed α- adrenergic blocking activity. Furthermore, the extract produced a cardio-selective response in the paired atria and decreased the blood pressure in anesthetized normotensive rats. The plant extract proved to be non-toxic in oral acute and chronic toxicity studies and did not demonstrate any sign of histopathological lesions. These results suggested that the plant extract was non-toxic and could be used in the management of lifetime therapies of respiratory and cardiovascular disorders without any unwanted effects.