Alpha-linolenic acid selectively inhibits the contraction of pig coronary arteries mediated through prostanoid TP receptors.

We examined the inhibitory effects of α-linolenic acid (ALA) on the contractions of pig coronary arteries. ALA concentration-dependently inhibited the contractions elicited by U46619 and prostaglandin F2α without affecting those elicited by 80 mM KCl, histamine, acetylcholine, and serotonin. ALA rightward shifted the concentration-response curve of U46619, and Schild plot analysis revealed that ALA competitively antagonized U46619. Furthermore, ALA inhibited the increase in intracellular Ca2+ concentration caused by TP receptor stimulation but not that caused by FP receptor stimulation. These results suggest that ALA behaves as a selective antagonist of TP receptors in coronary arteries.

Platelet-activating factor contracts guinea pig esophageal muscularis mucosae by stimulating extracellular Ca2+ influx through diltiazem-insensitive Ca2+ channels.

Platelet-activating factor (PAF) is expected to increase esophageal motility. However, to the best of our knowledge, this has not been examined. Thus, we investigated the contractile effects of PAF on guinea pig (GP) esophageal muscularis mucosae (EMM) and the extracellular Ca2+ influx pathways responsible. PAF (10-9-10-6 M) contracted EMM in a concentration-dependent manner. PAF (10-6 M)-induced contractions were almost completely suppressed by apafant (a PAF receptor antagonist, 3 × 10-5 M). In EMM strips, PAF receptor and PAF-synthesizing/degrading enzyme mRNAs were detected. PAF (10-6 M)-induced contractions were abolished by extracellular Ca2+ removal but were not affected by diltiazem [a voltage-dependent Ca2+ channel (VDCC) inhibitor, 10-5 M]. PAF (10-6 M)-induced contractions in the presence of diltiazem were significantly suppressed by LOE-908 [a receptor-operated Ca2+ channel (ROCC) inhibitor, 3 × 10-5 M], SKF-96365 [an ROCC and store-operated Ca2+ channel (SOCC) inhibitor, 3 × 10-5 M], and LOE-908 plus SKF-96365. Among the tested ROCC/SOCC-related mRNAs, Trpc3, Trpc6, and Trpv4/Orai1, Orai3, and Stim2 were abundantly expressed in EMM strips. These results indicate that PAF potently induces GP EMM contractions that are dependent on extracellular Ca2+ influx through ROCCs/SOCCs, and VDCCs are unlikely to be involved.

Inhibitory Actions of Antidepressants, Hypnotics, and Anxiolytics on Recombinant Human Acetylcholinesterase Activity.

Alzheimer's disease (AD) is accompanied by behavioral and psychological symptoms of dementia (BPSD), which is often alleviated by treatment with psychotropic drugs, such as antidepressants, hypnotics, and anxiolytics. If these drugs also inhibit acetylcholinesterase (AChE) activity, they may contribute to the suppression of AD progression by increasing brain acetylcholine concentrations. We tested the potential inhibitory effects of 31 antidepressants, 21 hypnotics, and 12 anxiolytics on recombinant human AChE (rhAChE) activity. At a concentration of 10-4 M, 22 antidepressants, 19 hypnotics, and 11 anxiolytics inhibited rhAChE activity by <20%, whereas nine antidepressants (clomipramine, amoxapine, setiptiline, nefazodone, paroxetine, sertraline, citalopram, escitalopram, and mirtazapine), two hypnotics (triazolam and brotizolam), and one anxiolytic (buspirone) inhibited rhAChE activity by ≥20%. Brotizolam (≥10-6 M) exhibited stronger inhibition of rhAChE activity than the other drugs, with its pIC50 value being 4.57 ± 0.02. The pIC50 values of the other drugs were <4, and they showed inhibitory activities toward rhAChE at the following concentrations: ≥3 × 10-6 M (sertraline and buspirone), ≥10-5 M (amoxapine, nefazodone, paroxetine, citalopram, escitalopram, mirtazapine, and triazolam), and ≥3 × 10-5 M (clomipramine and setiptiline). Among these drugs, only nefazodone inhibited rhAChE activity within the blood concentration range achievable at clinical doses. Therefore, nefazodone may not only improve the depressive symptoms of BPSD through its antidepressant actions but also slow the progression of cognitive symptoms of AD through its AChE inhibitory actions.

Platelet-activating factor (PAF) enhances guinea pig detrusor smooth muscle contractile activities by stimulating voltage-dependent Ca2+ channels and store-operated Ca2+ channels.

We investigated the extracellular Ca2+ influx pathways involved in platelet-activating factor (PAF)-enhanced guinea pig detrusor smooth muscle (DSM) contractile activities. One micromolar PAF-enhanced DSM contractile activities were completely inhibited by extracellular Ca2+ removal and strongly suppressed by voltage-dependent Ca2+ channel (VDCC) inhibitors. PAF-enhanced DSM contractile activities remaining in the presence of verapamil (10 µM) were not inhibited by LOE-908 (30 µM, an inhibitor of receptor-operated Ca2+ channels (ROCCs)), but were almost completely inhibited by SKF-96365 (30 µM, an inhibitor of store-operated Ca2+ channels (SOCCs) and ROCCs). These results suggest that VDCCs and SOCCs are responsible for PAF-enhanced DSM contractile activities.

Pharmacological study on the enhancing effects of U46619 on guinea pig urinary bladder smooth muscle contraction induced by acetylcholine and α,ß-methylene ATP and the possible involvement of protein kinase C.

We examined whether U46619 (a prostanoid TP receptor agonist) could enhance the contractions of guinea pig urinary bladder smooth muscle (UBSM) in response to acetylcholine (ACh) and an ATP analog (α,ß-methylene ATP (αß-MeATP)) through stimulation of the UBSM TP receptor and whether protein kinase C (PKC) is involved. U46619 (10-7 M) markedly enhanced UBSM contractions induced by electrical field stimulation and ACh/αß-MeATP (3 × 10-6 M each), the potentiation of which was completely suppressed by SQ 29,548 (a TP receptor antagonist, 6 × 10-7 M). PKC inhibitors did not attenuate the ACh-induced contractions enhanced by U46619 although they partly suppressed the U46619-enhanced, αß-MeATP-induced contractions. While phorbol 12-myristate 13-acetate (PMA, a PKC activator, 10-6 M) did not enhance ACh-induced contractions, it enhanced αß-MeATP-induced contractions, an effect that was completely suppressed by PKC inhibitors. αß-MeATP-induced contractions, both with and without U46619 enhancement, were strongly inhibited by diltiazem. U46619/PMA enhanced 50 mM KCl-induced contractions, the potentiation of which was partly/completely attenuated by PKC inhibitors. These findings suggest that U46619 potentiates parasympathetic nerve-associated UBSM contractions by stimulating UBSM TP receptors. PKC-increased Ca2+ influx through voltage-dependent Ca2+ channels may partially play a role in purinergic receptor-mediated UBSM contractions enhanced by TP receptor stimulation.

Pharmacological Studies on the Ca2+ Influx Pathways in Platelet-Activating Factor (PAF)-Induced Mouse Urinary Bladder Smooth Muscle Contraction.

Platelet-activating factor (PAF) not only acts as a mediator of platelet aggregation, inflammation, and allergy responses but also as a constrictor of various smooth muscle (SM) tissues, including gastrointestinal, tracheal/bronchial, and pregnancy uterine SMs. Previously, we reported that PAF induces basal tension increase (BTI) and oscillatory contraction (OC) in mouse urinary bladder SM (UBSM). In this study, we examined the Ca2+ influx pathways involved in PAF-induced BTI and OC in the mouse UBSM. PAF (10-6 M) induced BTI and OC in mouse UBSM. However, the PAF-induced BTI and OC were completely suppressed by extracellular Ca2+ removal. PAF-induced BTI and OC frequencies were markedly suppressed by voltage-dependent Ca2+ channel (VDCC) inhibitors (verapamil (10-5 M), diltiazem (10-5 M), and nifedipine (10-7 M)). However, these VDCC inhibitors had a minor effect on the PAF-induced OC amplitude. The PAF-induced OC amplitude in the presence of verapamil (10-5 M) was strongly suppressed by SKF-96365 (3 × 10-5 M), an inhibitor of receptor-operated Ca2+ channel (ROCC) and store-operated Ca2+ channel (SOCC), but not by LOE-908 (3 × 10-5 M) (an inhibitor of ROCC). Overall, PAF-induced BTI and OC in mouse UBSM depend on Ca2+ influx and the main Ca2+ influx pathways in PAF-induced BTI and OC may be VDCC and SOCC. Of note, VDCC may be involved in PAF-induced BTI and OC frequency, and SOCC might be involved in PAF-induced OC amplitude.

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.

Phenylephrine-Induced Contraction in Guinea Pig Thoracic Aorta Is Triggered by Stimulation of α1L-Adrenoceptors Functionally Coupled with Store-Operated Ca2+ Channels and Voltage-Dependent Ca2+ Channels.

We examined whether the α1L-adrenoceptor (AR), which shows low affinity (pA2 < 9) for prazosin (an α1-AR antagonist) and high affinity (pA2 ≈ 10) for tamsulosin/silodosin (α1A-AR antagonists), is involved in phenylephrine-induced contractions in the guinea pig (GP) thoracic aorta (TA). Intracellular signaling induced by α1L-AR activation was also examined by focusing on Ca2+ influx pathways. Tension changes of endothelium-denuded TAs were isometrically recorded and mRNA encoding α-ARs/Ca2+ channels and their related molecules were measured using RT-quantitative PCR. Phenylephrine-induced contractions were competitively inhibited by prazosin/tamsulosin, and their pA2 value were calculated to be 8.53/9.74, respectively. These contractions were also inhibited by silodosin concentration-dependently. However, the inhibition was not competitive fashion with the apparent pA2 value being 9.48. In contrast, phenylephrine-induced contractions were not substantially suppressed by L-765314 (an α1B-AR antagonist), BMY 7378 (an α1D-AR antagonist), yohimbine, and idazoxan (α2-AR antagonists). Phenylephrine-induced contractions were markedly inhibited by YM-254890 (a Gq protein inhibitor) or removal of extracellular Ca2+, and partially inhibited by verapamil (a voltage-dependent Ca2+ channel (VDCC) inhibitor). The residual contractions in the presence of verapamil were slightly inhibited by LOE 908 (a receptor-operated Ca2+ channel (ROCC) inhibitor) and strongly inhibited by SKF-96365 (a store-operated Ca2+ channel (SOCC) and ROCC inhibitor). Among the mRNA encoding α-ARs/SOCC-related molecules, α1A-AR (Adra1a)/Orai3, Orai1, and Stim2 were abundant in this tissue. In conclusion, phenylephrine-induced contractions in the GP TA can be triggered by stimulation of Gq protein-coupled α1L-AR, followed by activation of SOCCs and VDCCs.

Effects of NP-1815-PX, a P2X4 Receptor Antagonist, on Contractions in Guinea Pig Tracheal and Bronchial Smooth Muscles.

Administration of a P2X4 receptor antagonist to asthma model mice improved asthma symptoms, suggesting that P2X4 receptor antagonists may be new therapeutics for asthma. However, the effects of these antagonists on tracheal/bronchial smooth muscle (TSM and BSM) have not been investigated. This study examined the effects of NP-1815-PX, a selective P2X4 receptor antagonist, on guinea pig TSM and BSM contractions. In epithelium-intact TSM, NP-1815-PX (10-5 M) strongly suppressed ATP-induced contractions. ATP-induced contractions were strongly suppressed by indomethacin (3 × 10-6 M) and ONO-8130 (a prostanoid EP1 receptor antagonist, 10-7 M). ATP-induced contractions were partially suppressed by SQ 29,548 (a prostanoid TP receptor antagonist, 3 × 10-7 M), although the difference was not significant. In contrast, ATP-induced contractions were not affected by AL 8810 (a prostanoid FP receptor antagonist, 10-5 M) or L-798,106 (a prostanoid EP3 receptor antagonist, 10-8 M). NP-1815-PX (10-5-10-4 M) strongly suppressed U46619 (a TP receptor agonist)- and prostaglandin F2α (PGF2α)-induced epithelium-denuded TSM and BSM contractions, which were largely inhibited by SQ 29,548. Additionally, NP-1815-PX (10-5-10-4 M) strongly suppressed the U46619-induced increase in intracellular Ca2+ concentrations in human TP receptor-expressing cells. However, NP-1815-PX (10-4 M) did not substantially inhibit the TSM/BSM contractions induced by carbachol, histamine, neurokinin A, or 50 mM KCl. These findings indicate that NP-1815-PX inhibits guinea pig TSM and BSM contractions mediated through the TP receptor, in addition to the P2X4 receptor, whose stimulation mainly induces EP1 receptor-related mechanisms. Thus, these findings support the usefulness of NP-1815-PX as a therapeutic drug for asthma.

Docosahexaenoic Acid Selectively Suppresses U46619- and PGF2α-Induced Contractions in Guinea Pig Tracheal Smooth Muscles.

We investigated the potential inhibitory effects of docosahexaenoic acid (DHA) on the contractions of guinea pig tracheal smooth muscles in response to U46619 (a thromboxane A2 (TXA2) mimetic) and prostaglandin F2α (PGF2α) to examine whether this n-3 polyunsaturated fatty acid suppresses prostanoid-induced tracheal contractions. DHA (3 × 10-5 M) significantly suppressed tracheal contractions elicited by lower concentrations of U46619 (10-8 M) and PGF2α (5 × 10-7 M) (vs. control), although it did not suppress the contractions induced by higher concentrations (U46619: 10-7 M; PGF2α: 10-5 M). Supporting these findings, DHA (4 × 10-5 M/6 × 10-5 M) shifted the concentration-response curves for U46619 (10-9-10-6 M) and PGF2α (10-8-10-5 M) to the right. However, the slope of the regression line in the Schild plot of DHA vs. U46619/PGF2α was larger than unity. The tracheal contractions induced by U46619 (10-8 M) and PGF2α (5 × 10-7 M) were significantly suppressed by the prostanoid TP receptor antagonist SQ 29,548 (10-6 M) (vs. ethanol-treated). In contrast, DHA (4 × 10-5 M) did not show significant inhibitory effects on the contractions induced by acetylcholine (10-8-10-4 M), histamine (10-8-10-4 M), and leukotriene D4 (10-11-10-7 M) (vs. ethanol-treated). These findings indicate that DHA selectively suppresses tracheal contractions induced by U46619 and PGF2α. Therefore, DHA may be a useful therapeutic agent against asthma associated with tracheal/bronchial hyper-constriction caused by prostanoids including TXA2 and PGF2α.

Docosahexaenoic Acid and Eicosapentaenoic Acid Inhibit the Contractile Responses of the Guinea Pig Lower Gastrointestinal Tract.

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are n-3 polyunsaturated fatty acids (PUFAs), and are abundant in fish oil. These n-3 PUFAs have been reported to improve the lower gastrointestinal (LGI) disorders such as ulcerative colitis and Crohn's disease through their anti-inflammatory effects. However, there are few studies on the effect of n-3 PUFAs on motility of the LGI tract, such as the ileum and colon, the parts frequently affected by these inflammatory disorders. To elucidate the effects of DHA and EPA on the LGI tract motility, we performed comparative evaluation of their effects and linoleic acid (LA), an n-6 PUFA, on contractions in the ileal and colonic longitudinal smooth muscles (LSMs) isolated from guinea pigs. In the ileal and colonic LSMs, DHA and EPA (3 × 10-5 M each) significantly inhibited contractions induced by acetylcholine (ACh), histamine, and prostaglandin (PG) F2α (vs. control), and these effects are stronger than that of LA (3 × 10-5 M). In the colonic LSMs, DHA and EPA also significantly inhibited contractions induced by PGD2 (vs. control). In addition, DHA and EPA significantly inhibited CaCl2-induced ileal and colonic LSM contractions in Ca2+-free 80 mM-KCl solution (vs. control). Any ileal and colonic LSM contractions induced by ACh, histamine, PGF2α, and CaCl2 were completely suppressed by verapamil (10-5 M), a voltage-gated/dependent Ca2+ channel (VGCC/VDCC) inhibitor. These findings suggest that DHA and EPA could improve the abnormal contractile functions of the LGI tract associated with inflammatory diseases, partly through inhibition of VGCC/VDCC-dependent ileal and colonic LSM contractions.

Inhibitory Effects of Antipsychotics on the Contractile Response to Acetylcholine in Rat Urinary Bladder Smooth Muscles.

The clinical applications of antipsychotics for symptoms unrelated to schizophrenia, such as behavioral and psychological symptoms, in patients with Alzheimer's disease, and the likelihood of doctors prescribing antipsychotics for elderly people are increasing. In elderly people, drug-induced and aging-associated urinary disorders are likely to occur. The most significant factor causing drug-induced urinary disorders is a decrease in urinary bladder smooth muscle (UBSM) contraction induced by the anticholinergic action of therapeutics. However, the anticholinergic action-associated inhibitory effects of antipsychotics on UBSM contraction have not been sufficiently assessed. In this study, we examined 26 clinically available antipsychotics to determine the extent to which they inhibit acetylcholine (ACh)-induced contraction in rat UBSM to predict the drugs that should not be used by elderly people to avoid urinary disorders. Of the 26 antipsychotics, six (chlorpromazine, levomepromazine (phenothiazines), zotepine (a thiepine), olanzapine, quetiapine, clozapine (multi-acting receptor targeted antipsychotics (MARTAs))) competitively inhibited ACh-induced contractions at concentrations corresponding to clinically significant doses. Further, 11 antipsychotics (perphenazine, fluphenazine, prochlorperazine (phenothiazines), haloperidol, bromperidol, timiperone, spiperone (butyrophenones), pimozide (a diphenylbutylpiperidine), perospirone, blonanserin (serotonin-dopamine antagonists; SDAs), and asenapine (a MARTA)) significantly suppressed ACh-induced contraction; however, suppression occurred at concentrations substantially exceeding clinically achievable blood levels. The remaining nine antipsychotics (pipamperone (a butyrophenone), sulpiride, sultopride, tiapride, nemonapride (benzamides), risperidone, paliperidone (SDAs), aripiprazole, and brexpiprazole (dopamine partial agonists)) did not inhibit ACh-induced contractions at concentrations up to 10-5 M. These findings suggest that chlorpromazine, levomepromazine, zotepine, olanzapine, quetiapine, and clozapine should be avoided by elderly people with urinary disorders.

Effects of Catecholamine Metabolites on Beta-Adrenoceptor-Mediated Relaxation of Smooth Muscle: Evaluation in Mouse and Guinea-Pig Trachea and Rat Aorta.

The ß-adrenoceptor (ß-AR)-mediated pharmacological effects of catecholamine (CA) metabolites are not well known. We examined the effects of seven CA metabolites on smooth muscle relaxation in mouse and guinea pig (GP) tracheas and rat thoracic aorta. Among them, metadrenaline (MA) significantly relaxed GP trachea (ß2-AR dominant), even in the presence of clorgiline, a monoamine oxidase-A inhibitor. In mouse trachea (ß1-AR dominant), normetadrenaline (NMA) and MA (10-4 M each) apparently did not affect isoprenaline (ISO)-induced relaxation, but significantly inhibited it in the presence of clorgiline. ISO-induced relaxation was also unaffected by 3,4-dihydroxyphenylglycol (DHPG) (10-4 M), but significant suppression was observed with the addition of 3,5-dinitrocatechol, a catechol-O-methyltransferase inhibitor. In GP trachea, NMA, MA, 3,4-dihydroxymandelic acid (DOMA), and DHPG (10-4 M each) significantly augmented ISO-induced relaxation. However, in the presence of clorgiline plus 3,5-dinitrocatechol, both NMA and MA (10-4 M) significantly suppressed ISO-induced relaxation. DHPG (10-4 M) also significantly suppressed ISO-induced relaxation in the presence of 3,5-dinitrocatechol. In rat thoracic aorta, DHPG (10-4 M) significantly suppressed relaxation induced by CGP-12177 A (a ß3-AR partial agonist) in the presence of 3,5-dinitrocatechol plus propranolol. Our findings indicate that 1) MA may possess ß2-AR agonistic action; 2) NMA and MA augment ß2-AR-mediated tracheal relaxation in the absence of CA metabolic inhibitors, though themselves possessing ß1-, ß2-AR antagonistic action (ß2 > ß1); 3) DHPG exhibits ß1-, ß2-, ß3-AR antagonistic action, and this is particularly marked for ß3-AR. Our observations may help explain some of the pathologies associated with pheochromocytoma, which is characterized by increased CA metabolite levels.

Prehospital Epinephrine as a Potential Factor Associated with Prehospital Rearrest.

Objective: To investigate the impact of epinephrine on prehospital rearrest and re-attainment of prehospital return of spontaneous circulation (ROSC). Methods: Data for 9,292 (≥ 8 years) out-of-hospital cardiac arrest (OHCA) patients transported to hospitals by emergency medical services were collected in Ishikawa Prefecture, Japan during 2010-2018. Univariate and multivariable analyses were retrospectively performed for 1,163 patients with prehospital ROSC. Results: Of 1,163 patients, rearrest occurred in 272 (23.4%) but not in 891 (76.6%). Both single and multiple doses of epinephrine administered before prehospital ROSC (adjusted odds ratio (OR): 3.62, 95% confidence interval (CI): 2.42-5.46 for 1 mg, and 4.27, 2.58-6.79 for ≥ 2 mg) were main factors associated with rearrest. The association between initial and rearrest rhythms was significantly associated with epinephrine administration (p = 0.02). However, the rearrest rhythm was primarily associated with the initial rhythm (p < 0.01). The majority of patients with the non-shockable initial rhythm had pulseless electrical activity (PEA) as the rearrest rhythm, regardless of epinephrine administration (80.4% for administration, 81.6% for no administration). When the initial rhythm was shockable, the primary rearrest rhythms in patients with and without epinephrine administration before prehospital ROSC were PEA (52.2%) and ventricular fibrillation/pulseless ventricular tachycardia (56.8%), respectively. Only epinephrine administration after rearrest was associated with prehospital re-attainment of ROSC (adjusted OR: 2.49, 95% CI: 1.20-5.19). Stepwise multivariable logistic regression analyses revealed that neurologically favorable outcome was poorer in patients with rearrest than those without rearrest (9.9% vs. 25.0%, adjusted OR: 0.42, 95% CI: 0.23-0.73). The total prehospital doses of epinephrine were associated with poorer neurological outcome in a dose-dependent manner (adjusted OR: 0.22, 95% CI: 0.13-0.36 for 1 mg; 0.09, 0.04-0.19 for 2 mg; 0.03, 0.01-0.09 for ≥ 3 mg, no epinephrine as a reference). Transportation to hospitals with a unit for post-resuscitation care was associated with better neurological outcome (adjusted OR: 1.53, 95% CI: 1.02-2.32). Conclusions: The requirement for epinephrine administration before prehospital ROSC was associated with subsequent rearrest. Routine epinephrine administrations and rearrest were associated with poorer neurological outcome of OHCA patients with prehospital ROSC.

Sustainable Effects of Distigmine Bromide on Urinary Bladder Contractile Function.

Distigmine bromide (distigmine) is a reversible carbamate cholinesterase (ChE) inhibitor that is used to treat myasthenia gravis. In Japan, it is also used as a remedy for urination disorder (underactive bladder). The most distinctive pharmacological feature of distigmine is its long-lasting action compared to that of other ChE inhibitors. In animals and humans, distigmine was reported to inhibit acetylcholinesterase (AChE) and improve myasthenia gravis for an extended period. Few studies have examined the sustainability of this enhancing effect on the contractile function of urinary bladder smooth muscle. In addition, the cause of this long-lasting feature remains unclear. In this review, we present our findings for the long-lasting feature of distigmine on isolated urinary bladder contraction and in vivo urinary function of guinea pig. We also present our results on the mechanism of its long-lasting sustainability using recombinant human AChE.

Pharmacological Characteristics of Anxiolytics on Acetylcholine-Induced Contractions in Rat Detrusor Smooth Muscle.

INTRODUCTION: Benzodiazepine anxiolytics are believed to cause urination disorders due to their anticholinergic effects. OBJECTIVE: This study was carried out to investigate the potential inhibitory effects of 15 clinically available anxiolytics in Japan on acetylcholine (ACh)-induced contractions in rat detrusor smooth muscle (DSM) to predict whether these anxiolytics could induce urination disorders. METHODS: -Effects of anxiolytics on contractions induced by ACh and 80 mmol/L KCl solution in rat DSM and effects of anxiolytics on specific binding of [N-methyl-3H]scopolamine ([3H]NMS) in mouse cerebral cortex were investigated. RESULTS AND CONCLUSIONS: ACh-induced contractions in rat DSM were inhibited by clotiazepam and diazepam (benzodiazepine anxiolytics) at concentrations that were clinically relevant. These contractions were also significantly inhibited by paroxetine, escitalopram (selective serotonin reuptake inhibitors -[SSRIs]), and hydroxyzine (a histamine H1 receptor antagonist), albeit at concentrations that substantially exceeded clinically achievable blood levels. At a concentration of 10-5 mol/L, paroxetine, escitalopram, and hydroxyzine inhibited 80 mmol/L high-KCl solution-induced rat DSM contractions but not clotiazepam and diazepam. Paroxetine, escitalopram, and hydroxyzine also inhibited specific binding of [3H]NMS in mouse cerebral cortex but clotiazepam and diazepam did not. In contrast to the effects of the abovementioned anxiolytics, ACh-induced contractions were not significantly affected by tofisopam, alprazolam, lorazepam, bromazepam, oxazolam, chlordiazepoxide, clonazepam, ethyl loflazepate (benzodiazepine anxiolytics), fluvoxamine (an SSRI), or tandospirone (a serotonin 5-HT1A receptor agonist). These findings suggest that most clinically used anxiolytics are not likely to result in anticholinergic-induced urination disorders within their clinically achievable blood concentration ranges. However, clotiazepam and diazepam may induce urination disorders within their clinical dose ranges via nonanticholinergic inhibition of DSM contractility.

Characterization of binding of antipsychotics to muscarinic receptors using mouse cerebral cortex.

Antipsychotics are often the first-line treatment for behavioral and psychological symptoms of dementia. However, the potential anticholinergic effects of antipsychotics could counteract the therapeutic effects of cholinesterase inhibitors used to treat dementia. We investigated the inhibitory effects of 26 antipsychotics on [N-Methyl-3H]scopolamine specific binding in mouse cerebral cortex. At 10-5 M, chlorpromazine, levomepromazine, prochlorperazine, timiperone, zotepine, pimozide, blonanserin, olanzapine, quetiapine, and clozapine inhibited [N-Methyl-3H]scopolamine binding by > 45%. Furthermore, the pKi values of chlorpromazine, levomepromazine, zotepine, olanzapine, and clozapine overlapped with their clinically achievable blood concentrations. Therefore, the anticholinergic properties of these antipsychotics could attenuate the effects of cholinesterase inhibitors.

Effects of Distigmine on the Mechanical Activity of Urinary Bladder Smooth Muscle.

Distigmine bromide (distigmine) is a reversible carbamate cholinesterase (ChE) inhibitor. Its principle clinical application is in the treatment of myasthenia gravis. Distigmine is also used as a remedy for dysuria and glaucoma. Its effectiveness in the management of dysuria has been demonstrated in several clinical reports. Distigmine may improve (enhance) urinary bladder smooth muscle (UBSM) contraction during micturition by inhibiting acetylcholine (ACh) decomposition. However, the pharmacological effects of distigmine on UBSM have not been adequately studied so far. In this review article, we summarize the reported effects of distigmine on the contractile responses elicited by exogenous and endogenous ACh in isolated UBSM preparations. We also discuss the effects of distigmine on the UBSM basal tone and the contractile response of UBSM to ATP, which is co-released with ACh from parasympathetic nerve terminals.

Noradrenaline-Induced Relaxation of Urinary Bladder Smooth Muscle Is Primarily Triggered through the ß3-Adrenoceptor in Rats.

ß-Adrenoceptors are subclassified into 3 subtypes (ß1-ß3). Among these, ß3-adrenoceptors are present in various types of smooth muscle and are believed to play a role in relaxation responses of these muscles. ß3-Adrenoceptors are also present in urinary bladder smooth muscle (UBSM), although their expression varies depending on the animal species. To date, there has been little information available about the endogenous ligand that stimulates ß3-adrenoceptors to produce relaxation responses in UBSM. In this study, to determine whether noradrenaline is a ligand of UBSM ß3-adrenoceptors, noradrenaline-induced relaxation was analyzed pharmacologically using rat UBSM. We also assessed whether noradrenaline metabolites were ligands in UBSM. In isolated rat urinary bladder tissues, mRNAs for ß1-, ß2-, and ß3-adrenoceptors were detected using RT-PCR. In UBSM preparations contracted with methacholine (3 × 10-5 M), noradrenaline-induced relaxation was not inhibited by the following antagonists: atenolol (10-6 M; selective ß1-adrenoceptor antagonist), ICI-118,551 (3 × 10-8 M; selective ß2-adrenoceptor antagonist), propranolol (10-7 M; non-selective ß-adrenoceptor antagonist), and bupranolol (10-7 M; non-selective ß-adrenoceptor antagonist). In the presence of propranolol (10-6 M), noradrenaline-induced relaxation was competitively inhibited by bupranolol (3 × 10-7-3 × 10-6 M) or SR59230A (10-7-10-6 M; selective ß3-adrenoceptor antagonist), with their pA2 values calculated to be 6.64 and 7.27, respectively. None of the six noradrenaline metabolites produced significant relaxation of methacholine-contracted UBSM. These findings suggest that noradrenaline, but not its metabolites, is a ligand for ß3-adrenoceptors to produce relaxation responses of UBSM in rats.

Assessment of Inhibitory Effects of Hypnotics on Acetylcholine-Induced Contractions in Isolated Rat Urinary Bladder Smooth Muscle.

The present study aimed to investigate the potential inhibitory effects of 21 clinically available hypnotics on acetylcholine (ACh)-induced contractions in rat urinary bladder smooth muscle (UBSM) in order to predict whether these hypnotics could induce voiding impairment. ACh-induced contraction in rat UBSM was inhibited only by diphenhydramine (a histamine H1 receptor antagonist) at a concentration that was clinically relevant. ACh-induced contraction was also significantly inhibited by flurazepam (a benzodiazepine hypnotic) and suvorexant (an orexin receptor antagonist), albeit at concentrations that substantially exceeded clinically achievable blood levels. These three drugs (at 10-5 M) also inhibited high-KCl (80 mM) Locke-Ringer solution-induced contractions. In contrast to the effects of the abovementioned hypnotics, ACh-induced contractions were not significantly affected by triazolam, etizolam, brotizolam, lormetazepam, estazolam, flunitrazepam, nitrazepam (benzodiazepine hypnotics), thiopental, thiamylal, pentobarbital, amobarbital, secobarbital, phenobarbital (barbiturate hypnotics), zolpidem (an imidazopyridine hypnotic), zopiclone (a cyclopyrrolone hypnotic), ramelteon (a melatonin receptor agonist), bromovalerylurea, and chloral hydrate. These findings suggest that most clinically used hypnotics are not likely to result in anticholinergic-induced dysuria within their clinically achievable blood concentration ranges. Diphenhydramine may, however, induce voiding impairment, an action attributable to diminished UBSM contractility within its clinical dose range.