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We studied the inhibitory actions of docosahexaenoic acid (DHA) on the contractions induced by carbachol (CCh), angiotensin II (Ang II), and bradykinin (BK) in guinea pig (GP) gastric fundus smooth muscle (GFSM), particularly focusing on the possible inhibition of store-operated Ca 2+ channels (SOCCs). DHA significantly suppressed the contractions induced by CCh, Ang II, and BK; the inhibition of BK-induced contractions was the strongest. Although all contractions were greatly dependent on external Ca 2+ , more than 80% of BK-induced contractions remained even in the presence of verapamil, a voltage-dependent Ca 2+ channel inhibitor. BK-induced contractions in the presence of verapamil were not suppressed by LOE-908 (a receptor-operated Ca 2+ channel (ROCC) inhibitor) but were suppressed by SKF-96365 (an SOCC and ROCC inhibitor). BK-induced contractions in the presence of verapamil plus LOE-908 were strongly inhibited by DHA. Furthermore, DHA inhibited GFSM contractions induced by cyclopiazonic acid (CPA) in the presence of verapamil plus LOE-908 and inhibited the intracellular Ca 2+ increase due to Ca 2+ addition in CPA-treated 293T cells. These findings indicate that Ca 2+ influx through SOCCs plays a crucial role in BK-induced contraction in GP GFSM and that this inhibition by DHA is a new mechanism by which this fatty acid inhibits GFSM contractions., (© 2024. The Author(s).)

Platelet-activating factor (PAF), a phospholipid mediator, was discovered in 1972 as an inducer of platelet aggregation. Subsequent studies have revealed that PAF has a variety of biological functions, such as its role as a potent proinflammatory mediator. Additionally, PAF regulates the contractile functions of various types of smooth muscle (SM), such as the (1) endothelium-dependent relaxation of vascular SM; (2) contraction and epithelium-dependent relaxation of airway SM; (3) contraction of gastrointestinal SM; and (4) contraction of uterine SM, which occurs more strongly in pregnant females. PAF is produced in platelets, monocytes, neutrophils, and macrophages, which are cells related to thrombus formation and inflammation/immune responses. Furthermore, PAF is produced in various other cells throughout the body. Interestingly, recent studies have focused on the urinary bladder (UB) as a PAF-producing organ since the accumulation of this phospholipid is enhanced in patients with bladder cancer and interstitial cystitis/bladder pain syndrome, especially those who smoke. Therefore, in UB tissue, PAF may play a substantial role as an inducer or enhancer of cancers and inflammatory diseases. However, the effects of PAF on the immediate mechanical activities of UBSM have not been investigated to date. In this regard, we recently discovered that PAF strongly enhances mechanical activities (muscle tone and spontaneous contractile activity) in UBSM tissues isolated from guinea pigs and mice. In this review article, we present our data on these PAF effects together with the possible underlying mechanisms. We also discuss the potential pathophysiological roles of this phospholipid in UB diseases and disorders.

Since its first discovery as a bioactive phospholipid inducing potent platelet aggregation, platelet-activating factor (PAF) has been shown to be involved in a wide variety of inflammatory and allergic disease states. Many pharmacological studies in the 1980s and 1990s also showed that PAF induces endothelium-dependent vascular relaxation and contraction of various smooth muscles (SMs), including those in the airway, gastrointestinal organs, and uterus. However, since the late 1990s, there have been few reports on the SM contractions induced by PAF. The lower urinary tract (LUT), particularly the urinary bladder (UB) has attracted recent attention in SM pharmacology research because patients with LUT dysfunctions including overactive bladder are increasing as the population ages. In addition, recent clinical studies have implicated the substantial role of PAF in the inflammatory state in LUT because its production increases with smoking and with cancer. However, the effects of PAF on mechanical activities of LUT SMs including UBSM have not been investigated to date. Recently, we found that PAF very strongly increased mechanical activities of UBSM in guinea pigs and mice, and partly elucidated the possible mechanisms underlying these actions of PAF. In this review, we describe the effects of PAF on LUT SMs by introducing our recent findings obtained in isolated UBSMs and discuss the physiological and pathophysiological significance. We also introduce our data showing the effects of PAF on the SM mechanical activities of genital tissues (prostate and vas deferens).

In this study, we investigated the effects of platelet-activating factor (PAF) on the basal tone and spontaneous contractile activities of guinea pig (GP) and mouse urinary bladder (UB) smooth muscle (UBSM) tissues to determine whether PAF could induce UBSM tissue contraction. In addition, we examined the mRNA expression of the PAF receptor, PAF-synthesizing enzyme (lysophosphatidylcholine acyltransferase, LPCAT), and PAF-degrading enzyme (PAF acetylhydrolase, PAF-AH) in GP and mouse UB tissues using RT-qPCR. PAF (10 -9 -10 -6  M) strongly enhanced the basal tone and spontaneous contractile activities (amplitude and frequency) of GP and mouse UBSM tissues in a concentration-dependent manner. The enhancing effects of PAF (10 -6  M) on both GP and mouse UBSM contractile activities were strongly suppressed by pretreatment with apafant (a PAF receptor antagonist, GP: 10 -5  M; mouse: 3 × 10 -5  M). The PAF receptor (Ptafr), LPCAT (Lpcat1, Lpcat2), and PAF-AH (Pafah1b3, Pafah2) mRNAs were detected in GP and mouse UB tissues. These findings reveal that PAF strongly enhances the contractile mechanical activities of UBSM tissues through its receptor and suggest that the PAF-synthesizing and -degrading system exists in UBSM tissues. PAF may serve as both an endogenous UBSM constrictor and an endogenous mediator leading to detrusor overactivity., (© 2022. The Author(s).)

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 A 2 (TXA 2 ) mimetic) and prostaglandin F 2α (PGF 2α ) 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 PGF 2α (5 × 10 -7  M) (vs. control), although it did not suppress the contractions induced by higher concentrations (U46619: 10 -7  M; PGF 2α : 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 PGF 2α (10 -8 -10 -5  M) to the right. However, the slope of the regression line in the Schild plot of DHA vs. U46619/PGF 2α was larger than unity. The tracheal contractions induced by U46619 (10 -8  M) and PGF 2α (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 D 4 (10 -11 -10 -7  M) (vs. ethanol-treated). These findings indicate that DHA selectively suppresses tracheal contractions induced by U46619 and PGF 2α . Therefore, DHA may be a useful therapeutic agent against asthma associated with tracheal/bronchial hyper-constriction caused by prostanoids including TXA 2 and PGF 2α .

Aims: We examined the potential stimulatory effects of U46619 (a prostanoid TP receptor agonist) and five prostanoids on the contractile activities of urinary bladder smooth muscle (UBSM), focusing on the role of the TP receptor and its associated Ca 2+ influx routes to understand the roles of prostanoids in the regulation of UB contractile activity., Main Methods: Changes in the basal tone and spontaneous contractile activity (amplitude and frequency) of isolated guinea pig UBSM were measured isotonically. The presence of TP receptors in UBSM was examined by RT-qPCR and immunofluorescence., Key Findings: U46619, prostaglandin (PG) E 2 , PGF 2α , and PGA 2 enhanced UBSM basal tone and spontaneous contractile activities, which were measured as amplitudes and frequencies. The enhancing effects of U46619 were completely suppressed by SQ 29,548 (a TP receptor antagonist), which also partially suppressed the stimulating effects of other prostanoids. The expression of TP receptors in UBSMs was verified at the mRNA and protein level. The enhancing effects of U46619 completely disappeared in Ca 2+ -free solution. U46619-enhanced basal tone was completely suppressed by verapamil, an inhibitor of voltage-dependent Ca 2+ channels (VDCCs), and verapamil strongly decreased the spontaneous contraction frequency. The spontaneous contractions remaining in the presence of verapamil were strongly suppressed by SKF-96365 (an inhibitor of receptor-operated Ca 2+ channels (ROCCs)/store-operated Ca 2+ channels (SOCCs)), but not by LOE-908 (an inhibitor of ROCCs)., Significance: Prostanoids can enhance UBSM contractile activities and thus may be endogenous candidates for induction of detrusor overactivity. The TP receptor and TP-receptor-activated VDCCs/SOCCs are key molecules responsible for these effects., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Distigmine bromide (distigmine) is a carbamate cholinesterase (ChE) inhibitor, which is mainly used for the treatment of myasthenia gravis. Distigmine is also used in Japan for the treatment for underactive bladder and glaucoma. The effectiveness of distigmine for underactive bladder treatment has been confirmed by many clinical reports, and this effect is thought to be caused by potentiating urinary bladder smooth muscle contraction due to inhibition of acetylcholine degradation during micturition. However, the pharmacological effects of distigmine on urinary bladder smooth muscle have not been well studied. The most distinctive pharmacological feature of distigmine is that it shows long-lasting effects than other ChE inhibitors; however, few studies have investigated the persistence of the enhancing effect of distigmine on the contractile function of urinary bladder smooth muscle. Moreover, this mechanism remains unclear. In this review, we present our findings on the mechanism of the potentiating effect of distigmine on isolated guinea pig urinary bladder smooth muscle contraction. We also discuss the long-lasting potentiating effect of distigmine on urinary bladder motility and the mechanism of these effects using guinea pig urinary bladder smooth muscle in vivo and in vitro. In addition, we present our investigations on the long-lasting mechanism of distigmine using recombinant human acetylcholinesterase.

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.

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) F 2α (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 PGD 2 (vs. control). In addition, DHA and EPA significantly inhibited CaCl 2 -induced ileal and colonic LSM contractions in Ca 2+ -free 80 mM-KCl solution (vs. control). Any ileal and colonic LSM contractions induced by ACh, histamine, PGF 2α , and CaCl 2 were completely suppressed by verapamil (10 -5  M), a voltage-gated/dependent Ca 2+ 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.

Certain catecholamine metabolites exert significant pharmacological effects. Herein, we evaluated the pharmacological activities of catecholamine metabolites in the rat thoracic aorta, prostate, and spleen to determine whether these metabolites affect the contractile functions of smooth muscle tissue via direct action on α-adrenoceptors and α-adrenoceptor subtypes. Among the catecholamine metabolites examined, normetadrenaline and metadrenaline (10 -4  M each) produced relatively strong contractions in the rat thoracic aorta. Maximum aortic contractions induced by normetadrenaline (≈70% of phenylephrine (3 × 10 -7  M)-induced contractions) and metadrenaline (≈45%) were significantly smaller than those induced by phenylephrine (≈95%). Normetadrenaline and metadrenaline (10 -4  M each) inhibited phenylephrine (3 × 10 -7  M)-induced aortic contractions, which were not affected by propranolol (10 -6  M), by 5-20%. Normetadrenaline- and metadrenaline (3 × 10 -5  M each)-induced aortic contractions were strongly inhibited by prazosin (10 -8  M; an α 1 -adrenoceptor antagonist) and BMY 7378 (10 -8 -10 -7  M; a selective α 1D -adrenoceptor antagonist). Metadrenaline (3 × 10 -5  M)-induced aortic contractions were also significantly inhibited by silodosin (10 -9  M; a selective α 1A -adrenoceptor antagonist). Normetadrenaline and metadrenaline (3 × 10 -5  M each) caused silodosin (10 -9  M)-sensitive prostate contractions but did not cause a prominent spleen contraction. Maximum prostate contractions induced by metadrenaline (≈100% of phenylephrine (3 × 10 -5  M)-induced contractions) were nearly identical to those induced by phenylephrine (≈100%) but were significantly larger than those induced by normetadrenaline (≈80%). These findings suggest that normetadrenaline and metadrenaline act as a partial α 1D/ α 1A -adrenoceptor agonist and partial α 1D -adrenoceptor/full α 1A -adrenoceptor agonist, respectively, functioning as adrenaline system stabilizers in α 1D/ α 1A -adrenoceptor-abundant smooth muscle tissues., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., (© 2019 S. Karger AG, Basel.)

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., (© 2019 S. Karger AG, Basel.)

We investigated the potential augmenting effects of 19 clinically available antidepressants on noradrenaline (NA)-induced contractions in guinea pig urethra smooth muscle (USM). Concentration-response curves for NA-induced contractions in guinea pig USM strips were obtained in the absence or presence of selected antidepressants. Desipramine, an active metabolite of imipramine, produced a contraction and potentiated NA-induced contraction at the distal urethra without affecting the proximal urethra. Further, nortriptyline and amoxapine, tricyclic antidepressants, produced a contraction and potentiated NA-induced contraction at the distal urethra. NA-induced contraction was unaffected or reduced by imipramine, clomipramine, trimipramine, and amitriptyline at the proximal and distal urethra. Maprotiline, a tetracyclic antidepressant, potentiated NA-induced contraction at the distal urethra. NA-induced contraction was unaffected by mianserin at the proximal and distal urethra. Paroxetine, a selective serotonin reuptake inhibitor (SSRI), potentiated NA-induced contraction at the distal urethra, while NA-induced contraction was unaffected by fluvoxamine, sertraline, and escitalopram at the proximal and distal urethra. Milnacipran, a serotonin-noradrenaline reuptake inhibitor (SNRI), potentiated NA-induced contraction at the proximal and distal urethra, whereas duloxetine potentiated it at the distal urethra. Mirtazapine slightly inhibited NA-induced contraction at the distal urethra. Aripiprazole and sulpiride did not affect NA-induced contractions at the proximal nor distal urethra. Trazodone inhibited NA-induced contraction at both urethras. Desipramine, nortriptyline, amoxapine, maprotiline, paroxetine, milnacipran, and duloxetine likely induce urinary disturbance by increasing urethral resistance and augmenting NA-induced contraction, which should be carefully considered when delivering guidance for drug administration to patients., (© 2019 John Wiley & Sons Australia, Ltd.)

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., (© 2019 S. Karger AG, Basel.)

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 H 1 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.

Distigmine bromide (distigmine) is a reversible carbamate group cholinesterase (ChE) inhibitor. Although mainly used clinically for the treatment of myasthenia gravis, distigmine is also indicated for detrusor underactivity in Japan. According to the pharmacological classification of distigmine, its therapeutic effect against detrusor underactivity appears to be produced by enhanced urinary bladder smooth muscle (UBSM) contractility due to an increased concentration of acetylcholine between parasympathetic nerve endings and UBSM cells. However, ATP as well as acetylcholine is also released from parasympathetic nerve endings that dominate UBSM. The present study was thus carried out to investigate the potentiating effects of distigmine on the two UBSM contractile components in response to parasympathetic nerve stimulation induced by electrical field stimulation (EFS). In isolated guinea pig UBSM tissues, EFS (1-16Hz) produced tetrodotoxin-sensitive, frequency-dependent contractions. The contractile responses to EFS were largely diminished by atropine (10 -6 M), and the remaining contractile components in the presence of atropine were virtually abolished by α,β-methylene adenosine triphosphate (α,β-mATP) (10 -4 M). Distigmine (10 -6 M) significantly potentiated EFS-induced contractile components generated in the presence of α,β-mATP (10 -4 M), but did not potentiate EFS-induced contractile components generated in the presence of atropine (10 -6 M). These findings clearly indicate that distigmine strongly potentiates UBSM contraction selectively induced by parasympathetic nerve-derived acetylcholine, suggesting a potential mechanism by which distigmine restores detrusor underactivity., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Distigmine bromide (distigmine), a reversible carbamate cholinesterase inhibitor, is used in Japan for treating detrusor underactivity, myasthenia gravis, and glaucoma. Although there is clinical evidence about the effectiveness of distigmine in the treatment of detrusor underactivity, mechanisms by which distigmine restores impaired urinary bladder smooth muscle (UBSM) contractility have not been fully investigated. The aims of this study were to investigate the potentiating effects of distigmine on UBSM contractions in response to parasympathetic nerve stimulation induced by electrical field stimulation (EFS) in mice. In isolated mouse UBSM, EFS (1-16 Hz) produced tetrodotoxin-sensitive, frequency-dependent contractions. The contractile responses to EFS were largely attenuated by atropine (10-6 mol/l). UBSM contractility that occurred in the presence of atropine was nearly eliminated by the addition of α,β-methylene adenosine triphosphate (α,β-mATP, 10-4 mol/l). Distigmine (3 × 10-7 mol/l) significantly potentiated EFS-induced contractile responses engendered in the presence of α,β-mATP (10-4 mol/l), but not atropine (10-6 mol/l). These findings indicate that distigmine powerfully potentiates UBSM contractions selectively induced by parasympathetic nerve-derived acetylcholine, thereby demonstrating a potential mechanism by which it stimulates detrusor contractile function., (© 2016 S. Karger AG, Basel.)

Background/aims: To investigate the potential inhibitory effects of 18 clinically available antidepressants on acetylcholine (ACh)-induced contractions in guinea pig urinary bladder smooth muscle (UBSM) in order to predict whether they may induce voiding impairment., Methods: Concentration-response curves for ACh-induced contractions in guinea pig UBSM strips were obtained in the absence or presence of selected antidepressants. When inhibitory effects indicated competitive antagonism, pA2 values against ACh were calculated and compared to plausible antidepressant blood concentrations., Results: ACh-induced contraction was antagonized competitively within clinical dose ranges by tricyclic antidepressants (imipramine, amitriptyline, trimipramine, clomipramine, nortriptyline, and amoxapine), maprotiline (a tetracyclic antidepressant), and mirtazapine (a noradrenergic and specific serotonergic antidepressant). ACh-induced contraction was also significantly inhibited by mianserin (a tetracyclic antidepressant), paroxetine and sertraline (serotonin-selective reuptake inhibitors, SSRIs), and duloxetine (a serotonin noradrenaline (norepinephrine) reuptake inhibitor, SNRI), albeit at concentrations that substantially exceeded clinically achievable blood levels. However, ACh-induced contractions were not significantly affected by fluvoxamine and escitalopram (SSRIs), milnacipran (an SNRI), trazodone (a serotonin 5-HT2A receptor antagonist), sulpiride (a dopamine D2 receptor antagonist), or aripiprazole (a dopamine partial agonist)., Conclusion: These findings suggest that in addition to tricyclics, some relatively novel antidepressants such as mirtazapine can induce voiding impairment, attributed to diminished UBSM contractility from the inhibition of muscarinic receptors in the UBSM., (© 2016 S. Karger AG, Basel.)

Objective: The purpose of this study was to compare the multifidus electromyographic activity in healthy women during the performance of the same Pilates exercise under 2 conditions., Methods: Sixteen healthy, active, female practitioners of Pilates (mean age, 24.3; SD, 3.1 years) were assessed through surface electromyography during 2 Pilates exercises (swan dive and breast stroke). Muscle activity during the experimental tasks was recorded with 1 pair of electrodes placed bilaterally on the multifidi. Data were normalized to maximal activity recorded during dynamic activity., Results: The mean of the maximal isometric voluntary contraction was 662.4 N (SD, 195). In the phase comparison, the multifidus was the most recruited on the concentric phase during both exercises, ball (P=.001) and reformer (P=.001). In the comparison between conditions, the reformer presented more activation on swan dive (P=.04) and breast stroke (P=.001). The percentages of muscle activation varied between 50% and 60%, and the greatest activation was made on the reformer condition., Conclusion: The findings of this study show that Pilates exercises by healthy women, under different conditions, can cause changes to the multifidus muscle activation. The condition and the phase in which the muscle was more activated were the reformer and concentric phase, respectively, for both exercises. However, the results for the percentages of activation suggest that the intensity of recruitment may not be sufficient to strengthen muscle in healthy, trained subjects., (Copyright © 2013 National University of Health Sciences. Published by Mosby, Inc. All rights reserved.)

Inhibitory effects of docosahexaenoic acid (DHA) on blood vessel contractions induced by various constrictor stimulants were investigated in the rat thoracic aorta. The inhibitory effects of DHA were also compared with those of eicosapentaenoic acid (EPA) and linoleic acid (LA). DHA exhibited a strong inhibitory effect on the sustained contractions induced by U46619, a TXA(2) mimetic. This inhibitory effect of DHA was not affected by removal of the endothelium or by treatment with either indomethacin or N(ω)-nitro-l-arginine. DHA also significantly diminished PGF(2α)-induced contraction but did not show any appreciable inhibitory effects on the contractions to both phenylephrine (PE) and high-KCl. Similarly, EPA exhibited significant inhibitory effects against the contractions induced by both U46619 and PGF(2α) without substantially affecting either PE- or high-KCl-induced contractions. However, both DHA and EPA generated more potent inhibitions against contractions induced by U46619 than those by PGF(2α). In contrast, LA did not show significant inhibitory effects against any contractions, including those induced by U46619. The present findings suggest that DHA and EPA elicit more selective inhibition against blood vessel contractions that are mediated through stimulation of prostanoid receptors than those through α-adrenoceptor stimulation or membrane depolarization. Although DHA and EPA have similar inhibitory potencies against prostanoid receptor-mediated contractions, they had a more potent inhibition against TXA(2) receptor (TP receptor)-mediated contractions than against PGF(2α) receptor (FP receptor)-mediated responses. Selective inhibition by either DHA or EPA of prostanoid receptor-mediated blood vessel contractions may partly underlie the mechanisms by which these ω-3 polyunsaturated fatty acids exert their circulatory-protective effects.

The relationship between phosphorylation of 20,000 Da myosin light chain (MLC20) and contraction in response to mechanical stretch was investigated in the canine basilar artery. A slow stretch (at a rate of 1 mm/s and a stimulus period for 15 min) increased triphosphorylated MLC20 despite lowered intracellular calcium concentration and mechanical activities, such as myogenic tone, shortening velocity and stiffness of the artery. Nicardipine, a Ca2+ channel blocker, and ML-9, a myosin light chain kinase (MLCK) inhibitor, partially inhibited the stretch-induced MLC20 phosphorylation. The remained phosphorylation was further reduced by calphostin C, a protein kinase C (PKC) inhibitor. Y-27632, a Rho-kinase inhibitor, inhibited phosphorylation of myosin light chain phosphatase and attenuated MLC20 phosphorylation. These results suggest that slow stretch induces triphosphorylation of MLC20, which is mediated by MLCK, PKC, and Rho-kinase, and that the triphosphorylation of MLC20 does not result in myogenic contraction, rather seems to counteract it.

Blood vessels are always subjected to hemodynamic stresses including blood pressure and blood flow. The cerebral artery is particularly sensitive to hemodynamic stresses such as pressure and stretch, and shows contractions that are myogenic in nature; i.e., the mechanical response is generated by the vascular smooth muscle itself. The artery constricts in response to an increase in intraluminal pressure, and dilates in response to a decrease in the intraluminal pressure. We provide herein some insights into the mechanotransduction of vascular tissue; i.e., we discuss how the tissue is receptive to mechanical force and how the latter induces the specific signals leading to myogenic contraction in terms of mechanosensor action and subsequent intracellular signaling. The interactive role of tyrosine kinase, protein kinase C, and Rho/Rho-kinase systems in the mechanotransduction process is discussed, which systems also seem to play an important role in the development of experimental cerebral vasospasm. The study of the mechanotransduction in vascular tissue may aid in clarifying the mechanisms underlying vasospastic episodes and pathologic remodeling in cardiovascular diseases, and may potentially have therapeutic consequences.

The relationship between potentiation of serotonin (5-hydroxytryptamine, 5-HT)-induced contraction by endothelin-1 (ET-1) and the activity of protein kinase C (PKC) was examined in the porcine coronary artery. Low concentrations (30-100 pM) of ET-1 selectively potentiated the 5-HT-induced contraction 1.5 to 2 times over the control without any additional increase in myosin light-chain phosphorylation. The potentiation was attenuated by PKC down-regulation caused by phorbol 12-myristate 13-acetate. By Western blot analysis with isoform-specific antibodies to PKC, at least four isoforms (PKCalpha, PKCbeta1, PKCdelta and PKCzeta) were identified in the porcine coronary artery. PKCalpha and PKCdelta were mostly in the cytosol fraction, whereas PKCbeta1 and PKCzeta were almost equally distributed in the cytosol and membrane fractions in the resting and contractile states. Of the four isoforms, only PKCdelta was translocated from the cytosol to the membrane fraction during the contractile potentiation by ET-1. These results suggest that the activity of PKCdelta, a Ca2+-independent PKC isoform, is involved in the potentiation of 5-HT-induced contraction by ET-1 in the porcine coronary artery.

Phosphorylation of the myosin regulatory light chain (LC20-P1) is the major route of smooth muscle activation. However, after prior exposure to vanadate, permeabilized guinea pig taenia coli smooth muscle contracts in the absence of LC20-P1. We characterized the vanadate-induced contraction and investigated the mechanism of this novel activation pathway. Addition of vanadate to a control contracture (6.6 microM Ca2+) inhibits force (effective dose for 50% response was approximately 100 microM). In contrast, preincubation with high concentrations of vanadate (threshold at 1-2 mM) elicited a contraction on subsequent transfer of the fiber to a vanadate-free, Ca(2+)-free solution. Maximum isometric force of approximately 60% of control was obtained in fibers preincubated in 4 mM vanadate for 10 min. Addition of Ca2+ to a vanadate-induced contracture increased force, but the total force never exceeded the initial control. After maximal thiophosphorylation of LC20 with adenosine 5'-O-(3-thiotriphosphate), treatment with vanadate did not increase force. Unloaded shortening velocity (Vmax) was similar in Ca2+ and vanadate contractures and was additive. After thiophosphorylation, preincubation in vanadate had no effect on Vmax, suggesting that vanadate affected the number of activated bridges and not cycle rate. Vanadate mechanisms likely involve oxidation, since preincubation with 4 mM vanadate and 25 mM dithiothreitol (DTT) did not produce force. DTT could reverse a vanadate-induced contracture in 30-60 min. Subsequently, fibers demonstrated control contraction/relaxation cycles. Thus vanadate treatment did not cause irreversible damage, such as the extraction of proteins. Potential oxidation sites are proteins at 17 kDa and between 30 and 40 kDa, which were not alkylated by N-ethylmaleimide if they were treated in the presence of vanadate or in the rigor state. Vanadate-induced contractures are likely mediated by a reversible oxidation that activates cross bridges similarly to that of LC20-Pi and may play an important role in oxidant injury.

A method for the isolation of single smooth muscle cells from guinea pig taenia caeci is described. The single cells were prepared by digestion with 0.3% collagenase and 0.6% trypsin inhibitor in Ca2+-free solution. This procedure produced a high yield of intact cells. Most cells obtained by this procedure were relaxed and showed large contractile responses to excitatory stimulus. Maximal responses of the single cells to acetylcholine (ACh), histamine, and high K+ were attained within 20 sec, and the per cent decrease in cell length was 30-40%. Times for maximal responses of the single cells were shorter than those of the muscle strips. Single cells exhibited a dose-dependent graded response to calcium under depolarized conditions, ACh, histamine, and high K+, and a voltage- and duration-dependent response to electrical stimulation. The ED50s of ACh in the single cells and in the muscle strips were about 2 X 10(-8) and 1.5 X 10(-7) M, respectively. The muscle strips had a lower sensitivity than the single cells to ACh. The generally smooth surface of the relaxed cell contrasted with the numerous evaginations present on the fully contracted cell. I believe that single smooth muscle cells isolated from guinea pig using my technique are, at present, better for physiological and pharmacological studies than are cells isolated using other techniques.

1. Relationship between length-tension relation and phosphorylation of 20,000 dalton myosin light chain (LC20) in guinea-pig taenia caeci was investigated. 2. At in situ length (Lb), a good linear correlation was obtained between isometric tension and LC20 phosphorylation in high-K+-stimulated muscle. 3. In 100 mM K+-stimulated muscle, the active tension decreased at muscle lengths other than Lb, but no significant decrease in degree of LC20 phosphorylation was observed. 4. These results suggest that in guinea-pig taenia caeci, the major portion of the decrease in active tension at muscle lengths other than Lb is not due to a decrease in degree of activation.

The effects of isoproterenol on isometric force, unloaded shortening velocity, and myosin phosphorylation were examined in thin muscle bundles (0.1-0.2 mm diam) dissected from lamb tracheal smooth muscle. Methacholine (10(-6) M) induced rapid increases in isometric force and in phosphorylation of the 20,000-Da myosin light chain. Myosin phosphorylation remained elevated during steady-state maintenance of isometric force. The shortening velocity peaked at 15 s after stimulation with methacholine and then declined to approximately 45% of the maximal value by 3 min. Isoproterenol pretreatment inhibited methacholine-stimulated myosin light chain phosphorylation, shortening velocity, and force during the early stages of force generation. However, the inhibitory effect of isoproterenol on force and myosin phosphorylation is proportionally greater than that on shortening velocity. Isoproterenol pretreatment also caused a rightward non-parallel shift in the methacholine dose-response curves for both isometric tension and myosin light chain phosphorylation. These data demonstrate that isoproterenol attenuates the contractile properties of airway smooth muscles by affecting the rate and extent of myosin light chain phosphorylation, perhaps through a mechanism that involves the synergistic interaction of myosin light chain kinase phosphorylation and Ca2+ metabolism.

1. The effects of nicorandil on guinea-pig taenia caeci were investigated with the use of isolated smooth muscle cells and glycerin-treated muscle fiber bundles. 2. Nicorandil inhibited high K-, Ca2+- and carbachol-induced contractions in a dose-dependent manner without affecting 45Ca fluxes in isolated cells. 3. Nicorandil had no effect on ATP-induced contraction of glycerin-treated muscle fiber bundles. 4. The present results suggest that nicorandil may inhibit the contraction by action on the contractile proteins in an indirect manner in guinea-pig taenia caeci.

The present study was undertaken to investigate the role of cyclic AMP and cyclic GMP in the regulation of the contraction-relaxation cycle in isolated smooth muscle cells from guinea pig taenia caeci. The contraction of isolated smooth muscle cells induced by both caffeine (4 X 10(-3) M) and carbachol (10(-4) M) consisted of an initial activation phase and a following spontaneous relaxation phase. Isoproterenol (10(-4) M) inhibited the carbachol-induced contraction and raised intracellular cyclic AMP level of isolated smooth muscle cells. Both the inhibition of the carbachol-induced contraction and the rise in cyclic AMP level elicited by isoproterenol were blocked by propranolol (10(-4) M). Caffeine increased intracellular cyclic AMP level significantly and contracted isolated smooth muscle cells. Caffeine and carbachol contracted isolated smooth muscle cells and increased intracellular cyclic GMP level. Cyclic GMP was significantly increased in spontaneous relaxation phase. The data suggest that beta-adrenergic inhibitory effect on cholinergic excitation in the mammalian isolated smooth muscle cells is mediated by the change in intracellular cyclic AMP level and that cyclic GMP related to the relaxation in these single cells. Thus, it appears that caffeine-induced contraction might be due to the stimulation of Ca2+-induced Ca2+ release by cyclic AMP.

Effect of transverse tubule-disruption (T-disruption) on 14C-caffeine influx was studied using small bundles, consisting of 40 to 50 fibers, of frog semitendinosus muscle. The peak tension of caffeine contracture was inhibited by about 69% at 5 mM caffeine and by about 31% at 7 mM caffeine by T-disruption. In addition, the amount of caffeine influx in a small bundle was inhibited by about 52% at 5 mM caffeine and by about 28% at 7 mM caffeine by T-disruption, corresponding to the inhibition of the peak tension of caffeine contracture by T-disruption. These findings support our previous suggestion that caffeine enters the muscle fibers through the surface membrane and transverse tubuler membrane (T-membrane), and that the T-membrane contributes to the entry of caffeine more greatly at 5 mM caffeine than at 7 mM caffeine. Moreover, at 5 mM caffeine it was noted that there is a slight difference between the extent of inhibition of the caffeine influx and that of the peak contracture tension. From these results, it could be concluded that the inhibition of peak tension of caffeine contracture by T-disruption is mainly due to the inhibition of caffeine influx which occurs through the T-membrane and is partly due to the functional disturbance of the sarcoplasmic reticulum which is probably induced by T-disruption.

The effects of ruthenium red (RR) on electrical and mechanical responses, and on Ca fluxes at rest and during potassium contracture (K contracture) in frog skeletal muscle were examined. By the treatment of single fibers with RR, the peak tensions of twitch and K contracture were inhibited, the degree of inhibition being dependent on the concentration of RR and on the duration of pretreatment. The inhibitory effect of RR on K contracture was pronounced at lower concentrations of K. The recovery of twitch tension from complete inhibition was observed several minutes after removal of RR by washing the fiber in normal Ringer solution, and was accelerated by brief exposure to 190 mM K solution. Caffeine contracture was not affected by RR. Moreover, resting and action potentials were not changed by RR except for an enhancement of a negative afterpotential. From these results, it is concluded that RR blocks excitation-contraction coupling (E-C coupling) in frog skeletal muscle. In addition, Ca influx, especially early rapid Ca influx, was markedly inhibited by RR, although the amount of self-exchangeable Ca was not significantly decreased. On the basis of these results, the sites and mechanism of RR action were discussed.

The effects of okadaic acid, a phosphoprotein phosphatase inhibitor, on the contractile response and on myosin light chain phosphorylation were studied in intact lamb tracheal smooth muscle. The effects of okadaic acid were compared to the response of the same fibers stimulated with 1 microM methacholine, a concentration that induces 90% of maximal force. Okadaic acid (50 microM) produced a slow but maximal contraction that was accompanied by an increase in phosphorylation of the 20 kDa light chain of myosin. The myosin light chain phosphorylation pattern induced by okadaic acid, however, differed from that induced by methacholine. Ca2+ depletion, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin antagonist and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), a protein kinase C inhibitor, blocked or attenuated methacholine-induced contractions but had no significant effect on force development or myosin light chain phosphorylation induced by okadaic acid. These results suggest that phosphorylation of the 20 kDa light chain of myosin is essential for smooth muscle contraction; they also suggest that okadaic acid either uncovers or activates an apparently Ca2+ and calmodulin-independent protein kinase activity that phosphorylates the 20 kDa light chain of myosin at multiple sites.