EFS-induced Contractions Research Articles

Onvansertib, a polo-like kinase 1 inhibitor, inhibits prostate stromal cell growth and prostate smooth muscle contraction, which is additive to inhibition by α1-blockers

Prostate smooth muscle contraction and prostate enlargement contribute to lower urinary tract symptoms suggestive of benign prostatic hyperplasia. Recent evidence demonstrated that inhibitors for polo-like kinases (PLKs) inhibit smooth muscle contraction of human prostate tissues. However, their additive effects to α1-blockers, and effects on prostate growth are unknown. Here, we examined effects of a novel and highly selective PLK1 inhibitor, onvansertib on prostate smooth muscle contraction alone and in combination with α1-blockers, and on proliferation and viability of prostate stromal cells (WPMY-1). Prostate tissues were obtained from radical prostatectomy. Contractions were studied in an organ bath. Proliferation and viability were assessed by plate colony, EdU, and CCK-8 assay. Electric field stimulation (EFS)-induced contractions of human prostate tissues were inhibited to 34% by 100 nM and 1 μM onvansertib at 32 Hz, and to 48% and 47% by the α1-blockers tamsulosin and silodosin. Combination of onvansertib with tamsulosin or silodosin further reduced EFS-induced contractions in comparison to α1-blockers alone (59% and 61% respectively), and to onvansertib alone (68% for both). Noradrenaline-, phenylephrine-, methoxamine-, endothelin-1-, and ATP-induced contractions were inhibited by onvansertib (100 nM) to similar extent. Viability and proliferation of WPMY-1 cells were reduced in a concentration- and time-dependent manner (24–72 h, 10–100 nM). Onvansertib inhibits neurogenic, adrenergic, and endothelin-1- and ATP-induced contractions of human prostate smooth muscle, and proliferation of stromal cells. Contractions are reduced not more than 50% by α1-blockers. Combination of α1-blockers with onvansertib provides additive inhibition of prostate contractions.

Carvacrol acts as a potent selective antagonist of different types of nicotinic acetylcholine receptors and enhances the effect of monepantel in the parasitic nematode Ascaris suum

The neuromuscular system of parasitic nematodes has proven to be an efficient pharmacological target for antihelmintics. Some of the most frequently used antiparasitic drugs are agonists or antagonists of nicotinic acetylcholine receptors (nAChRs). The antinematodal mechanism of action of carvacrol involves the inhibition of parasite muscle contraction. We have examined the interaction of carvacrol with antinematodal drugs that are agonists of different subtypes of nAChRs and monepantel, which is a non-competitive antagonist of this receptor in A. suum. Additionally, we investigated the effect of carvacrol on the muscle type of nAChRs in the mammalian host. As orthosteric agonists of nAChR, pyrantel, morantel and befinijum lead to dose-dependent contractions of the neuromuscular preparation of Ascaris suum. Carvacrol 100 μM decreased the Emax of pyrantel, morantel and bephenium by 29%, 39% and 12 %, 39 % and 12 % respectively. The EC50 ratio was 3.43, 2.95 and 2.47 for pyrantel, morantel and bephinium, respectively. Carvacrol 300 u μM reduces the Emax of pyrantel, morantel and bephenium by 71%, 80% and 75 %, 80 % and 75 % respectively. The EC50 ratio for pyrantel, morantel and bephenium was 3.88, 3.19 and 4.83 respectively. Furthermore, carvacrol enhances the inhibitory effect of monepantel on A. suum contractions, which may have an effective clinical application. On the other hand, tested concentrations of carvacrol did not significantly affect the EFS-induced contractions of the rat diaphragm, indicating a lack of interaction with the postsynaptic nAChR at the muscle end plate in mammals, but the highest concentration (300 μM) caused a clear tetanic fade. Carvacrol exhibited a time and dose-dependent effect on the Rota-rod performances of rats with a high value of the ED50 (421.6 mg/kg). In our research, carvacrol dominantly exhibited characteristics of a non-competitive antagonist of nAChR in A. suum, and enhances the inhibitory effect of monepantel. The combination of monepantel and carvacrol may be clinically very effective, and the carvacrol molecule itself can be used as a promising platform for the development of new anthelmintic drugs.

Electrical field stimulation induces endothelium-dependent contraction of human umbilical cord vessels

Electrical field stimulation (EFS) has been used for decades in classical pharmacological preparations in order to characterize the mediators released by neural endings involved in smooth muscle contraction or relaxation. Since most of the human umbilical cord has no innervation, EFS has never been used in this preparation. This study aimed to investigate the effect of EFS on vascular responsiveness from human umbilical cord. Segments of the human umbilical cord were obtained from normotensive parturients and the human umbilical artery (HUA) and the human umbilical vein (HUV) were isolated and mounted in organ bath chambers. Electrical field stimulation-induced contractions in both HUA (2.35 ± 1.31 mN and 3.77 ± 2.31 mN for 8 Hz and 16 Hz respectively, n = 24) and HUV (3.81 ± 2.54 mN and 6.26 ± 4.51 mN for 8 Hz and 16 Hz respectively, n = 25). The addition of tetrodotoxin (1 μM) did not alter the EFS-induced contractions in both tissues (n = 5). Pre-incubation with atropine (10 and 100 μM), glibenclamide (10 μM) and indomethacin (10 μM) did not affect the EFS-induced contractions in both tissues. The contractions of both vessels were significantly reduced by pre-incubation of the tissues with phentolamine (10 and 100 μM). The endothelium removal almost abolished the EFS- induced contractions in both vessels (n = 5). In sandwich preparation, donor tissue (with endothelium) released a factor (s) that promoted contraction of the recipient tissue (endothelium removal) in both HUA and HUV (n = 5, respectively). Our findings indicate a potential role of endothelium-derived catecholamines in modulating HUA and HUV reactivities.

The basal release of endothelium-derived catecholamines regulates the contractions of Chelonoidis carbonaria aorta caused by electrical-field stimulation.

ABSTRACTThe contractions of Chelonoidis carbonaria aortic rings induced by electrical field stimulation (EFS) are not inhibited by blockade of the voltage-gated sodium channels by tetrodotoxin but almost abolished by the α1/α2-adrenoceptor antagonist phentolamine. The objective of this study was to identify the mediator(s) responsible for the EFS-induced contractions of Chelonoidis carbonaria aortic rings. Each ring was suspended between two wire hooks and mounted in isolated 10 ml organ chambers filled with oxygenated and heated Krebs-Henseleit's solution. Dopamine, noradrenaline and adrenaline concentrations were analysed by liquid chromatography coupled to tandem mass spectrometry. The contractions caused by dopamine and EFS were done in absence and presence of the nitric oxide (NO) synthesis inhibitor L-NAME, the NO-sensitive guanylyl cyclase inhibitor ODQ, the D1-like receptor antagonist SCH-23390, the D2-like receptor antagonists risperidone, quetiapine, haloperidol, and the tyrosine hydroxylase inhibitors salsolinol and 3-iodo-L-tyrosine. Basal concentrations of dopamine, noradrenaline and adrenaline were detected in Krebs-Henseleit solution containing the aortic rings. The catecholamine concentrations were significantly reduced in endothelium-denuded aortic rings. L-NAME and ODQ significantly potentiated the dopamine-induced contractions. The D2-like receptor antagonists inhibited the EFS-induced contractions of the aortic rings treated with L-NAME, whereas SCH 23390 had no effect. Similar results were observed in the contractions induced by dopamine in L-NAME treated aortic rings. These results indicate that catecholamines released by endothelium regulate the EFS-induced contractions. This may constitute a suitable mechanism by which reptilia modulate specific organ blood flow distribution.This paper has an associated First Person interview with the first author of the article.

The STK16 inhibitor STK16-IN-1 inhibits non-adrenergic and non-neurogenic smooth muscle contractions in the human prostate and the human male detrusor.

Mixed lower urinary tract symptoms (LUTS) (voiding symptoms suggestive of benign prostatic hyperplasia plus storage symptoms, which can be caused by overactive bladder) are common in men. Unwanted contraction of prostate and/or bladder smooth muscle has been implied in the pathophysiology of male LUTS. Here, we examined effects of the serine/threonine kinase 16 (STK16) inhibitor STK16-IN-1 on contraction of human tissues from the prostate and male detrusor. Tissues were obtained from radical prostatectomy and radical cystectomy. Contractions were studied in an organ bath and STK16 expressions by Western blot analyses and fluorescence staining. In prostate tissues, STK16-IN-1 (1μM) inhibited contractions induced by endothelin-1 and the thromboxane A2 analog U46619. Contractions of prostate tissues induced by noradrenaline, the α1-agonists phenylephrine and methoxamine, or electric field stimulation (EFS) were not changed by STK16-IN-1. In male detrusor tissues, STK16-IN-1 inhibited contractions induced by the cholinergic agonists carbachol and metacholine, and contractions induced by U46619. EFS-induced contractions of detrusor tissues were not changed by STK16-IN-1. Western blot analyses of prostate and detrusor tissues revealed bands matching the molecular weight of STK16. Fluorescence staining of prostate tissues using STK16 antibodies resulted in immunoreactivity in smooth muscle cells.STK16-IN-1 selectively inhibits non-adrenergic/non-neurogenic smooth muscle contractions in the male prostate and to limited extent in the bladder. Because non-adrenergic contractions in the male LUTS may account for limited efficacy of α1-blockers and for α1-blocker-resistant symptoms, studies assessing add-on of STK16-IN-1 to α1-blockers in mixed LUTS appear feasible.

Relaxing effects of clenbuterol, ritodrine, salbutamol and fenoterol on the contractions of horse isolated bronchi induced by different stimuli

β2-adrenoceptor agonists are considered the most effective drugs to counteract bronchoconstriction in horses with asthma, but only clenbuterol is commonly employed in clinical practice. We evaluated the effects of different selective β2 agonists: clenbuterol, ritodrine, salbutamol, and fenoterol on the contractions of isolated bronchial muscle of horses induced by electrical field stimulation (EFS), carbachol, histamine, and KCl. All β2 agonists reduced the amplitude of contraction induced by the different stimuli but with variable efficacy and potency. Fenoterol and salbutamol were more effective than clenbuterol in relaxing the bronchial contractions induced by EFS and histamine, and were able to completely abolish carbachol-induced contractions, unlike clenbuterol and ritodrine. The respective potency values (pEC50) of clenbuterol, ritodrine, salbutamol, and fenoterol were 7.74 ± 0.20, 7.77 ± 0.17, 7.30 ± 0.23, 8.01 ± 0.13, for EFS-induced contractions; 8.39 ± 0.26, 5.49 ± 0.28, 6.63 ± 0.14, 7.68 ± 0.11, for carbachol-induced contraction; 7.39 ± 0.27, 7.04 ± 0.28, 6.45 ± 0.34, 7.34 ± 0.22, for histamine-induced contraction; 7.15 ± 0.06, 6.07 ± 0.20, 6.48 ± 0.14, 6.70 ± 0.18, for KCl-induced contraction. Salbutamol and fenoterol showed a higher efficacy than clenbuterol in relaxing horse bronchial muscle pre-contracted by most stimuli. Clenbuterol displayed a good potency but a rather low efficacy, and this may be due to its partial agonist nature; ritodrine showed lower or not significantly different efficacy and potency compared to the other agonists. An evaluation of the clinical efficacy by fenoterol and salbutamol in horses with asthma could be of great interest to assess if they could represent more effective bronchodilators compared to clenbuterol.

Differences between young and aged rats in voiding frequency and detrusor muscle serotonergic contraction.

The involvement of serotonin (5-HT) in increased lower urinary tract symptoms in aging is unclear. We sought to compare voiding function and 5-HT induced detrusor contraction between young and aged rats. This study used young (2- to 3-month-old) and aged (26- to 30-month-old) male Fischer 344 rats. 1. Rats were housed in individual metabolic cages, and then the total volume of urination, volume per micturition, voiding frequency, and voiding interval were analyzed. 2. Using urinary bladder body strips, developed tension was recorded after cumulative addition of 5-HT (1-100 nM) in the absence or presence of tetrodotoxin (1 μM), and in the presence of tetrodotoxin with ketanserin (0.3-3 μM) or naftopidil (1 and 3 μM). We examined the effects of atropine, ketanserin, and naftopidil on electrical field stimulation (EFS)-induced contraction. 1. Compared to young rats, aged rats exhibited decreased voiding frequency and increased volume per micturition, but total volume of urination (normalized to body weight) did not differ. Moreover, voiding interval was significantly prolonged in aged rats during the active period. 2. In the presence of tetrodotoxin, pEC50 of 5-HT were significantly lower in aged rats than in young rats (P < 0.01), but the maximal response to 5-HT was not altered in the aged bladder. Ketanserin inhibited 5-HT-induced contraction in both groups, while suppression by naftopidil was relatively limited, especially in aged rats. EFS induced neurogenic contraction in a frequency-dependent manner. Atropine-resistant contraction was not inhibited by naftopidil, but was potentiated by ketanserin. Urination intervals were extended in aged rats, indicating that urination rhythm changed. In the senescent rat bladder, 5-HT induced detrusor contraction, but the effect of 5-HT and the naftopidil-sensitive contractile force were weaker than those in young rats. Additionally, 5-HT did not contribute to the increase in atropine-resistant EFS-induced contractions in aged rats.

Sildenafil corrects the increased contractility of rat detrusor muscle induced by alprostadil in vitro

Sildenafil (PDE5-inhibitor) and alprostadil (PGE1) are used in combination clinically for the management of some cases of erectile dysfunction. Despite the roles of prostaglandins (PG) and nitric oxide (NO) pathways in contractility of bladder smooth muscle are frequently studied, the effect of sildenafil/alprostadil combination and the crosstalk between NO/cGMP and PG pathways on bladder activity is not documented. Organ-bath experiments were performed using isolated rat detrusor muscle. Direct and neurogenic contractions were induced using ACh and electric stimulation (EFS, 4Hz, 80V, 1ms), respectively. The contractile responses in absence and presence of the tested drugs at different concentrations were compared. Results are expressed as mean ± SEM (n = 5-7). Alprostadil (0.01-10 μM) concentration-dependently potentiated ACh (100μM)- and EFS (4 Hz)- induced contraction. Maximum potentiation of ACh-contraction in presence of alprostadil was 40 ± 5%. Sildenafil potentiated ACh-induced contraction at low concentrations (0.01-1 μM), but inhibited it at higher ones (10-100 μM). IBMX (non-selective PDE-inhibitor, 0.01-100μM) and SNP (NO-donor, 1nM-1 mM) produced the same biphasic pattern. The potentiatory phase of sildenafil was inhibited by atropine (0.1μM), L-NAME (non-selective NOS-inhibitor, 100μM), N-PLA (nNOS-inhibitor, 30μM) or MB (nonselective GC-inhibitor, 10μM). In presence of sildenafil (0.1μM), the concentration-response curve of alprostadil (0.01-10μM) on both ACh and EFS-induced contraction was clearly shifted downward. A crosstalk between PGE1 and NO/cGMP pathways may exist. At low concentrations only, the effect of sildenafil on bladder contractility is dependent on NO/cGMP. cGMP intracellularly-elevated by sildenafil, may inhibit the activity of PLC and hence the cascade of EP1-receptors, thus masking the hyperactivity of bladder caused by alprostadil, which adds to the advantages of this combination.

Chronic Treatment With Acetylcholinesterase Inhibitors Attenuates Vascular Dysfunction in Spontaneously Hypertensive Rats.

Acetylcholinesterase inhibition prevents autonomic imbalance, reduces inflammation, and attenuates the development of hypertension. Considering that vascular dysfunction is a crucial feature of arterial hypertension, we investigated the effects of chronic administration of acetylcholinesterase inhibitors-pyridostigmine or donepezil-on vascular reactivity of spontaneously hypertensive rats (SHR). Endothelium-dependent relaxant responses to acetylcholine (ACh) and contractile responses induced by electric field stimulation (EFS) and alpha-adrenergic agonist were studied in mesenteric resistance arteries from SHR and Wistar Kyoto rats. SHR were treated for 16 weeks with vehicle, pyridostigmine (1.5 mg/kg/day) or donepezil (1.4 mg/kg/day). Pyridostigmine and donepezil decreased the vasoconstrictor responses to EFS, which were increased in vehicle-treated SHR. Acetylcholinesterase inhibition increased the modulatory effects of nitric oxide (NO) on SHR vascular reactivity, that is, N(ω)-nitro-(L)-arginine methyl ester (L-NAME) increased EFS-induced contractions and reduced ACh-induced relaxation, with more significant effects in pyridostigmine- and donepezil-treated SHR. The acetylcholinesterase inhibitors also decreased vascular reactive oxygen species levels. This study demonstrates for the first time that long-term administration of acetylcholinesterase inhibitors, pyridostigmine or donepezil, attenuates vascular reactivity dysfunction in SHR by decreasing reactive oxygen species generation and increasing NO bioavailability; possibly via increased endothelial NO synthase activity, and inhibition of NADPH oxidase activity.

Amiloride Relaxes Rat Corpus Cavernosum Relaxation In Vitro and Increases Intracavernous Pressure In Vivo

IntroductionThe antihypertensive effects of thiazide diuretics such as hydrochlorothiazide are commonly associated with erectile dysfunction. The association of hydrochlorothiazide/amiloride is not associated with erectile dysfunction. The hypothesis is that amiloride has beneficial effect in penile erection and, therefore, counterbalances the hydrochlorothiazide-induced disruptive effect. AimTo investigate the effects of amiloride and its analogues hexamethylamiloride and benzamil on rat isolated corpus cavernosa (CC) and intracavernous pressure (ICP) in anaesthetized rats. MethodsRat isolated CC were incubated with amiloride, hexamethylamiloride, and benzamil (10 and 100 μmol/L each), followed by phenylephrine, potassium chloride, and electrical field stimulation (EFS). Their effect on the relaxant responses to EFS and sodium nitroprusside were also determined. Oral (30 mg/kg) and intraperitoneal (3 mg/kg) treatments with amiloride were also investigated on nerve-evoked ICP. Main Outcome MeasuresIn vitro functional studies and in vivo ICP measurement on rat CC were performed. Additionally, phosphodiesterase type V isoform A1 activity and the mRNA expressions of Na+/H+ pump, epithelial sodium channel exchangers (ENaC) channels (α-, β- and γ subunits) and Na+/Ca2+ exchangers were evaluated in CC tissues. ResultsAmiloride and its analogues significantly reduced the phenylephrine-, potassium chloride–, and EFS-induced CC contractions, which were not changed by nitro-L-arginine methyl ester (100 μmol/L) or indomethacin (6 μmol/L). In phenylephrine-precontracted CC tissues, amiloride itself caused concentration-dependent relaxation and significantly increased the EFS-induced relaxation. Oral and intraperitoneal treatment with amiloride significantly increased the ICP. Phosphodiesterase type V isoform A1 activity was not affected by amiloride. Na+/H+ pump, ENaC, and Na+/Ca2+ exchanger mRNA expressions were all detected in rat CC tissues. Clinical ImplicationAmiloride analogues may have therapeutic potential for erectile dysfunction. Strength & LimitationsThe interesting effect of amiloride in penile erection was observed in both in vitro and in vivo methods. The evidence at the moment is restricted to rat CC. ConclusionAmiloride reduces in vitro CC contractility and enhances erectile function after oral and intraperitoneal administration, possibly via inhibition of ENaC.Campos R, Claudino MA, de Oliveira MG, et al. Amiloride Relaxes Rat Corpus Cavernosum Relaxation In Vitro and Increases Intracavernous Pressure In Vivo. J Sex Med 2019;16:500–511.

Evaluation of Antidepressant Effects on Recovery of Electrical Field Stimulation-Induced Contractions that have been Suppressed by Clonidine in Isolated Rat Vas Deferens

Background: A report examining whether clinically available antidepressants increase urethral smooth muscle contraction via antagonistic effects on the α2-adrenoceptor (α2-AR) is lacking. Objectives: The present study was performed to evaluate the potential of clinically available antidepressants to reverse α2-AR-mediated contractile inhibition in rat vas deferens, in order to predict whether they can induce voiding impairment. Method: The effects of 18 antidepressants of different classes on electrical field stimulation (EFS)-induced contractions suppressed by 10–8 mol/L clonidine (a selective α2-AR agonist) in isolated rat vas deferens were investigated and related to their respective clinical blood concentrations. Results: The EFS-induced contractions suppressed by clonidine were recovered by amitriptyline (a tricyclic antidepressant), mirtazapine (a noradrenergic and specific serotonergic antidepressant), and trazodone (a serotonin 5-HT2A receptor antagonist) at concentrations close to the clinical blood levels. EFS-induced contractions were also recovered by trimipramine, clomipramine (tricyclic antidepressants), mianserin (a tetracyclic antidepressant), sertraline (a selective serotonin reuptake inhibitor [SSRI]), and sulpiride (a dopamine D2-receptor antagonist), albeit at concentrations that substantially exceeded their clinically-achievable blood levels. EFS-induced contractions were not significantly affected by imipramine, nortriptyline, amoxapine (tricyclic antidepressants), maprotiline (a tetracyclic antidepressant), fluvoxamine, paroxetine, escitalopram (SSRIs), milnacipran, duloxetine (serotonin and noradrenaline reuptake inhibitors), and aripiprazole (a dopamine partial agonist). Conclusions: These findings suggest that amitriptyline, mirtazapine, and trazodone induce voiding impairment caused by increased urethral resistance by enhancing sympathetic nerve activities attributed to α2-AR antagonism.

Effect of pregnancy on the cholinergic responses of the bladder: role of acetylcholinesterase.

Pregnancy is associated with many functional changes of the urinary bladder. It was reported that most of healthy women complain from urinary symptoms during pregnancy. The parasympathetic system is mainly mediating bladder emptying. The aim of the study is to investigate the cholinergic effect and the role of acetylcholinesterase in the bladder during pregnancy. Sixteen rats were used in the present study as control group (non-pregnant) and pregnant group (18-20days pregnant). Isolated urinary smooth muscle strips were suspended in organ baths filled with Krebs' solution for isometric tension recording. Electric field stimulation (EFS), (0.1-40Hz), of the control and pregnant bladder preparations produced frequency-dependent contractions. Atropine (1µM) inhibited EFS-induced contractions in the two groups by 65% and 50% respectively indicating the response of cholinergic innervation. Neostigmine significantly enhanced EFS responses, confirming its selectivity for inhibiting acetylcholinesterase which is responsible for termination of acetylcholine. Concentration-response curves for acetylcholine were reduced in pregnant group than control. Concentration-response curves for ATP were increased in pregnant group than control. Neostigmine augmented concentration-response curves for acetylcholine in control and pregnant groups. The effect of neostigmine on acetylcholine contractile responses in pregnancy group was higher than in control. Urinary bladder dysfunction during pregnancy might be due to augmentation of acetylcholinesterase effect. This will lead to the decrease in response to cholinergic stimuli. New pharmaceutical drugs specifically affecting the enzyme in the bladder can help in avoiding the unpleasant urinary symptoms during pregnancy.

Electrical field-induced contractions on Crotalus durissus terrificus and Bothrops jararaca aortae are caused by endothelium-derived catecholamine.

Endothelium is the main source of catecholamine release in the electrical-field stimulation (EFS)–induced aortic contractions of the non- venomous snake Panterophis guttatus. However, adrenergic vasomotor control in venomous snakes such as Crotalus durissus terrificus and Bothrops jararaca has not yet been investigated. Crotalus and Bothrops aortic rings were mounted in an organ bath system. EFS-induced aortae contractions were performed in the presence and absence of guanethidine (30 μM), phentolamine (10 μM) or tetrodotoxin (1 μM). Frequency-induced contractions were also performed in aortae with endothelium removed. Immunohistochemical localization of both tyrosine hydroxylase (TH) and S-100 protein in snake aortic rings and brains, as well as in human tissue (paraganglioma tumour) were carried out. EFS (4 to 16 Hz) induced frequency-dependent aortic contractions in both Crotalus and Bothrops. The EFS-induced contractions were significantly reduced in the presence of either guanethidine or phentolamine in both snakes (p<0.05), whereas tetrodotoxin had no effect in either. Removal of the endothelium abolished the EFS-induced contractions in both snakes aortae (p<0.05). Immunohistochemistry revealed TH localization in endothelium of both snake aortae and human vessels. Nerve fibers were not observed in either snake aortae. In contrast, both TH and S100 protein were observed in snake brains and human tissue. Vascular endothelium is the main source of catecholamine release in EFS-induced contractions in Crotalus and Bothrops aortae. Human endothelial cells also expressed TH, indicating that endothelium- derived catecholamines possibly occur in mammalian vessels.

Αlpha1B-adrenoceptor-mediated positive inotropic and positive chronotropic actions in the mouse atrium

Modulation of cardiac contractility by α-adrenoceptor is well known in several mammals. Mice are useful experimental animals, but α-adrenoceptor-mediated responses have been examined only in the ventricles. To determine function of α-adrenoceptors in the atrium, effects of α-adrenoceptor agonists on spontaneous contraction and electrical-field stimulation (EFS)-induced contraction were examined. In addition, expression of α1A, α1B, α1D and β1-adrenoceptor mRNAs were examined. In the right atrium, noradrenaline and phenylephrine caused positive inotropic and positive chronotropic actions. However, methoxamine, clonidine and xylazine caused positive inotropic actions, but contractile frequency was decreased at high concentrations. Phenylephrine-induced positive inotropic and chronotropic actions were partially decreased by propranolol, and both actions remained in the presence of propranolol were inhibited by phentolamine or prazosin. A low concentration of silodosin (<100 nM) did not but a high concentration (1 μM) decreased the phenylephrine-induced chronotropic actions. Negative chronotropic actions of clonidine and xylazine were insensitive to propranolol and phentolamine. The EFS-induced contraction of the left atrium was potentiated by noradrenaline, phenylephrine and methoxamine but was not changed by clonidine or xylazine. Propranolol partially decreased the actions of phenylephrine, and prazosin caused additional inhibition. Expression of β1-, α1A-, α1B- and α1D-adrenoceptor mRNAs was found in the atrium, and the expression level of β1-adrenoceptor was the highest. Of α1-adrenoceptors, the expression level of α1B was higher than that of α1A and α1D. In conclusion, α1B-adrenoceptors are expressed in the mouse atrium and mediate both positive chronotropic and inotropic actions. In contrast, the α2-adrenoceptor is not functional in the isolated atrium.

Inhibition of Prostate Smooth Muscle Contraction by Inhibitors of Polo-Like Kinases.

Background: Prostate smooth muscle contraction plays an important role for pathophysiology and treatment of male lower urinary tract symptoms (LUTS) but is incompletely understood. Because the efficacy of available medication is limited, novel options and improved understanding of prostate smooth muscle contraction are of high demand. Recently, a possible role of polo-like kinase 1 (PLK1) has been suggested for smooth muscle contraction outside the lower urinary tract. Here, we examined effects of PLK inhibitors on contraction of human prostate tissue.Methods: Prostate tissues were obtained from radical prostatectomy. RT-PCR, Western blot and immunofluorescence were performed to detect PLK expression and phosphorylated PLK. Smooth muscle contractions were induced by electric field stimulation (EFS), α1-agonists, endothelin-1, or the thromboxane A2 analog U46619 in organ bath.Results: RT-PCR, Western blot, and immunofluorescence suggested expression of PLK1 in the human prostate, which may be located and active in smooth muscle cells. EFS-induced contractions of prostate strips were reduced by SBE 13 (1 μM), cyclapolin 9 (3 μM), TAK 960 (100 nM), and Ro 3280 (100 nM). SBE 13 and cyclapolin 9 inhibited contractions by the α1-agonists methoxamine, phenylephrine, and noradrenaline. In contrast, no effects of SBE 13 or cyclapolin 9 on endothelin-1- or U46619-induced contractions were observed.Conclusion: Alpha1-adrenergic smooth muscle contraction in the human prostate can be inhibited by PLK inhibitors. PLK-dependent signaling may be a new pathway, which promotes α1-adrenergic contraction of prostate smooth muscle cells. As contractions by endothelin and U46619 are not susceptible to PLK inhibition, this reflects divergent regulation of adrenergic and non-adrenergic prostate smooth muscle contraction.

Electrical field stimulation-induced contractions on Pantherophis guttatus corpora cavernosa and aortae.

A tetrodotoxin (TTX)-resistant mechanism is responsible for the electrical field stimulation (EFS)-induced contractions and relaxations of Crotalus durissus terrificus corpora cavernosa. Here it was investigated whether this mechanism also occurs in corpora cavernosa and aortae of the non-venomous snake Pantherophis guttatus corpora cavernosa and aortae. Corpora cavernosa and aortic rings isolated from Pantherophis guttatus snake were mounted in organ bath system for isometric tension recording. EFS-induced contractions in both tissues were performed in the presence and absence of guanethidine (30 μM), phentolamine (10 μM) and tetrodotoxin (1 μM). In another set of experiments, the endothelium was removed from aortic rings and EFS-induced contractions were performed in the denuded rings. Electrical field stimulation-induced contractions were frequency-dependent in Pantherophis guttatus corpora cavernosa and aortic rings. The contractions were significantly reduced in the presence of guanethidine (30 μM) or phentolamine (10 μM). Pre-treatment with tetrodotoxin had no effect on the EFS-induced contractions of either corpora cavernosa or aortic rings. Surprisingly, the EFS-induced contractions of aortic rings denuded of endothelium were almost abolished. These results indicate that the TTX-resistant mechanism is present in EFS-induced contractions of Pantherophis guttatus corpora cavernosa and aortae. The experiments performed in the aorta indicate that the endothelium is the main source for the release of catecholamines induced by EFS.

The novel mechanism of bitter taste receptors attenuating rat ventricular contractility

BackgroundBitter taste receptors (TAS2Rs) are a family of G‐protein coupled receptors, firstly identified in the tongue. Recent advances show the expression of TAS2Rs in extra‐oral organs, such as trachea, gastrointestinal tract, and heart. These findings raised up the interest of studying the physiological functions of TAS2Rs besides taste sensing. It has been demonstrated that the activation of TAS2Rs activates gustducin and release Gβγ, which leads to the inhibition of L‐type Ca2+ channels and the consequent airway smooth muscle relaxation.AimThis study investigates the expression of TAS2Rs in rat heart, and the mechanism of bitter compounds regulation of the contractility of cardiac muscle.MethodsRats were excised by thoracotomy and left ventricle strips were isolated. The strips were processed to study the expression levels of TAS2Rs mRNA by real time quantitative PCR (qPCR). Langendorff isolated heart models were used to record the left ventricle pressure and heart rate. Isometric tension recordings were used to measure electrical field stimulation (EFS,1 Hz – 5 V) and epinephrine‐induced contractions in combination with bitter compounds, denatonium and quinine. The conventional whole‐cell patch clamp techniques were used to record the voltage activated L‐type Ca2+ channel currents. The statistic data are expressed as mean ± SEM.ResultsWe detected the mRNA of 6 bitter receptors, TAS2R108, 120, 121, 126, 135, and 143 in the left ventricle tissues and isolated cardiomyocytes of rat, in which the mRNA level of TAS2R120 is the highest among the six followed by TAS2R143 and 121.In isolated perfused rat hearts denatonium (50 μM) reduced the heart rate from 235 ± 3 to 101 ± 12 (N=4). In isolated left ventricle strips, EFS and epinephrine induced phasic contractions which were inhibited completely by tetracaine (100 μM), a ryanodine channel blocker. Denatonium and quinine inhibited EFS‐induced ventricular contractions amplitude and frequency in a concentration dependent manner (N=6, n=6). Both of denatonium and quinine, at concentrations of 10 to 200 μM, caused a complete inhibition of epinephrine (10 μM)‐elicited phasic contractions (N=6, n=6).Nifedipine (10 μM), a selective L‐type Ca2+ channel blocker, partially inhibited EFS‐induced contractions, and the subsequent addition of denatonium (200 μM) and quinine (200 μM) cause a further and complete inhibition of the contractions (N=5, n=5). The non‐selective β‐adrenergic receptor antagonist, propranolol, did not have any effect on the EFS‐induced contractions. In isolated ventricular cardiomyocytes, denatonium (10 μM) reduced the L‐type Ca2+ channel currents density at 10 mV from 4.0 pA/pF to 1.6 pA/pF.ConclusionBitter taste receptor agonists, denatonium and quinine, induce negative ionotropic effects in rat hearts. Our data provide mechanistic evidence that TAS2Rs agonists can inhibit the cardiac contractions by attenuating the L‐type Ca2+ channels, and the consequent Ca2+ release by ryanodine receptors. TAS2Rs agonists can be useful pharmacological tools for the treatment of ventricular tachycardia.Support or Funding InformationSupported by Southwest University and the International Affair Agency of ChongqingThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Functional Impairment in the Corpus Cavernosum Related to a High Fat Diet is Prevented in Toll‐Like Receptor 9 Mutant Mice

High fat diets lead to obesity and trigger inflammatory processes which are associated with an increase in the toll‐like receptor 9 (TLR9) signaling pathway in rodent models of non‐alcoholic fatty liver disease. Furthermore, TLR9 signaling plays a role in inflammation regulation of adipose tissue in obesity and metabolic syndrome. Erectile dysfunction (ED) has been correlated with increased inflammatory mediators. Since obesity induces ED, we hypothesize that the impaired function in the corpus cavernosum may be related to an increase in the TLR9 signaling in obese mice. The aim of this study was to evaluate the relaxation and contractile responses of the corpus cavernosum of wild type and TLR9 mutant mice made obese by a high fat diet. Four week old male C57BL/6 and TLR9 mutant mice were fed a high fat diet for 12 weeks. The corpus cavernosum was then mounted on strip myograph for functional evaluation. The contractile and relaxation responses of the corpus cavernosum were evaluated by electrical field stimulation (EFS) and concentration response curves to acetylcholine and phenylephrine. We observed that the relaxation induced by EFS was decreased in wild type obese mice. Deletion of TLR9 prevented this decrease. On the other hand, EFS‐induced contraction at 8 Hz was higher in the corpus cavernosum of obese wild type mice. However, this increase was prevented in TLR9 mutant mice. Furthermore, we observed that the contraction induced by KCl solution (120 mM) and maximal contraction induced by phenylephrine tended to be increased in wild type obese mice only. No changes were observed in the relaxation induced by acetylcholine or in the potency of phenylephrine. In conclusion, these data suggest that TLR9 signaling pathway contributes to erectile dysfunction induced by a high fat diet.Support or Funding InformationNIH: NHLBI001961FAPESP: 2016/20592‐8This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Increased Tone of the Human Colon Muscle by Bisacodyl In Vitro.

Background/AimsAlthough bisacodyl is a widely administered laxative, its underlying mechanism of action remains generally unknown. This study focuses on investigating the effects of bisacodyl on the human colon muscle contraction, and elucidating its mechanism of action.MethodsSigmoid colon muscle strips (20 longitudinal and 18 circular muscles) were obtained from 20 subjects who underwent colectomy for colon cancer. Isometric force measurements were calculated in response to electrical field stimulation (EFS, 0.3 milliseconds in trains of 10 Hz for 20 seconds, 150 V). Peak and nadir (tone) during and after EFS, were measured in a controlled state, and after sequential addition of bisacodyl (1 μM), atropine (1 μM), N-nitro-L-arginine (L-NNA, 100 μM), MRS2500 (1 μM), and tetrodotoxin (TTX, 1 μM) to the organ bath.ResultsTransient phasic contractions were observed during EFS, and after cessation of EFS. In the longitudinal muscles, nadir during EFS, and tone after EFS, significantly increased after addition of bisacodyl, and persisted after sequential addition of atropine, L-NNA, MRS2500, and TTX, indicating a direct action of bisacodyl on the smooth muscle. In the second experiment, pretreatment of TTX abolished EFS-induced phasic contractions. Although no phasic contraction was produced after perfusion of bisacodyl, tone was increased, thereby supporting evidence of a direct mechanism of action of bisacodyl on the colon smooth muscle.ConclusionsBisacodyl increases the tone of longitudinal muscle in the human sigmoid colon through a direct action on the smooth muscle. Further study is warranted to investigate the neural mechanism of action of bisacodyl.

CAMP Catalyzing Phosphodiesterases Control Cholinergic Muscular Activity But Their Inhibition Does Not Enhance 5-HT4 Receptor-Mediated Facilitation of Cholinergic Contractions in the Murine Gastrointestinal Tract.

Background: As the signal transduction of 5-HT4 receptors on cholinergic neurons innervating smooth muscle is controlled by phosphodiesterase (PDE) 4 in porcine stomach and colon, and human large intestine, the in vivo gastroprokinetic effects of a 5-HT4 receptor agonist might be enhanced by combination with a selective PDE4 inhibitor. The presence of 5-HT4 receptors on cholinergic neurons towards murine gastrointestinal circular muscle was recently shown. If the control of this receptor pathway by PDE4 is also present in mice, this might be a good model for in vivo testing of the combination therapy. Therefore this study investigates the role of cAMP catalyzing PDEs in smooth muscle cell activity and in the intraneuronal signal transduction of the 5-HT4 receptors in the gastrointestinal tract of C57Bl/6J mice.Methods: In circular smooth muscle strips from murine fundus, jejunum, and colon, submaximal cholinergic contractions were induced by either electrical field stimulation (EFS) or by carbachol (muscarinic receptor agonist). The influence of the PDE inhibitors IBMX (non-selective), vinpocetine (PDE1), EHNA (PDE2), cilostamide (PDE3), and rolipram (PDE4) was tested on these contractions and on the facilitating effect of a submaximal concentration of prucalopride (5-HT4 receptor agonist) on EFS-induced contractions.Results: In the three gastrointestinal regions, IBMX and cilostamide concentration-dependently decreased carbachol- as well as EFS-induced contractions. Some inhibitory effect was also observed with rolipram. In the fundus a non-significant trend for an enhancement of the facilitating effect of prucalopride on EFS-induced contractions was observed with IBMX, but none of the selective PDE inhibitors enhanced the facilitating effect of prucalopride in fundus, jejunum or colon.Conclusion: In analogy with the porcine gastrointestinal tract, in murine fundus, jejunum, and colon circular smooth muscle PDE3 is the main regulator of the cAMP turnover, with some contribution of PDE4. In contrast to the porcine gastrointestinal tract, the in vitro facilitation of electrically induced cholinergic contractions by 5-HT4 receptor stimulation could not be enhanced by specific PDE inhibition. The C57Bl/6J murine model is thus not suitable for in vivo testing of a 5-HT4 receptor agonist combined with a selective PDE4 inhibitor.