Search

Two species of rice have been independently domesticated from different ancestral wild species in Asia and Africa. Comparison of mutations that underlie phenotypic and physiological alterations associated with domestication traits in these species gives insights into the domestication history of rice in both regions. Asian cultivated rice, Oryza sativa, and African cultivated rice, Oryza glaberrima , have been modified and improved for common traits beneficial for humans, including erect plant architecture, nonshattering seeds, nonpigmented pericarp, and lack of awns. Independent mutations in orthologous genes associated with these traits have been documented in the two cultivated species. Contrary to this prevailing model, selection for awnlessness targeted different genes in O. sativa and O. glaberrima . We identify Regulator of Awn Elongation 3 ( RAE3 ) a gene that encodes an E3 ubiquitin ligase and is responsible for the awnless phenotype only in O. glaberrima . A 48-bp deletion may disrupt the substrate recognition domain in RAE3 and diminish awn elongation. Sequencing analysis demonstrated low nucleotide diversity in a ~600-kb region around the derived rae3 allele on chromosome 6 in O. glaberrima compared with its wild progenitor. Identification of RAE3 sheds light on the molecular mechanism underlying awn development and provides an example of how selection on different genes can confer the same domestication phenotype in Asian and African rice.

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.

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.

Saccharomyces cerevisiae cells activate the Rim101 pathway to adapt to alkaline and salt stresses. Upon activation of this pathway, the transcription factor Rim101 undergoes proteolytic activation and regulates the expression of responsive genes. We found Rim101 to be a short-lived protein with a half-life of approximately 15 min. Its rapid turnover was supposedly mediated by the ubiquitin-proteasome system. Excess accumulation of the processed active Rim101 through its overexpression conferred tolerance to both alkaline and salt stresses in yeast cells; in contrast, it had detrimental effects under cadmium stress condition. Cadmium ion inhibited proteolytic activation of Rim101, implying reciprocal interaction between the Rim101 pathway and cadmium stress. Our results demonstrated yeast cells to be equipped with two protective systems to prevent overaccumulation of the processed active Rim101; Rim101 processing is inhibited when Rim101 level is high, and turnover of processed Rim101 is accelerated when it is abundant. Collectively, the results highlighted the flexible aspect of stress response in yeast cell; the cells not only prevent excess activation of one stress-responsive pathway but also facilitate its attenuation to cope with other environmental stresses.

This study was performed to elucidate whether eicosapentaenoic acid (EPA) suppresses spasm-prone blood vessel contractions induced by a thromboxane mimetic (U46619) and prostaglandin F2α (PGF2α) and determine whether the primary target of EPA is the prostanoid TP receptor. Accordingly, we assessed: (1) the tension changes in porcine basilar and coronary arteries, and (2) changes in the Fura-2 (an intracellular Ca2+ indicator) fluorescence intensity ratio at 510 nm elicited by 340/380 nm excitation (F340/380) in 293T cells expressing the human TP receptor (TP-293T cells) and those expressing the human prostanoid FP receptor (FP-293T cells). EPA inhibited both porcine basilar and coronary artery contractions induced by U46619 and PGF2α in a concentration-dependent manner, but it did not affect the contractions induced by 80 mM KCl. EPA also inhibited the increase in F340/380 induced by U46619 and PGF2α in TP-293T cells. In contrast, EPA showed only a marginal effect on the increase in F340/380 induced by PGF2α in FP-293T cells. These findings indicate that EPA strongly suppresses the porcine basilar and coronary artery contractions mediated by TP receptor and that inhibition of TP receptors partly underlies the EPA-induced inhibitory effects on these arterial contractions.

The polytopic plasma membrane protein Rim21 senses both the elevation of ambient pH and alterations in plasma membrane lipid asymmetry in the Rim101 pathway in budding yeast. Rim21 is known to undergo N-glycosylation, but the site and function of the glycosylation modification is not known. Using a systematic mutation analysis, we found that Rim21 is N-glycosylated at an unconventional motif located in the N-terminal extracellular region. The Rim21 mutant protein that failed to receive N-glycosylation showed prolonged protein lifetime compared to that of WT Rim21 protein. Although both the WT and mutant Rim21 localized to the plasma membrane, they exhibited different biochemical fractionation profiles. The mutant Rim21, but not WT Rim21, was mainly fractionated into the heavy membrane fraction. Further, compared to WT Rim21, mutant Rim21 was more easily solubilized with digitonin but was conversely more resistant to solubilization with Triton X-100. Despite these different biochemical properties from WT Rim21, mutant Rim21 protein could still activate the Rim101 pathway in response to external alkalization. Collectively, N-glycosylation of Rim21 is not indispensable for its activity as a sensor protein, but modulates the residence of Rim21 protein to some microdomains within the plasma membrane with distinct lipid conditions, thereby affecting its turnover.Key words: plasma membrane, lipid asymmetry, N-linked glycosylation, microdomain, Saccharomyces cerevisiae.

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.

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. Keywords: Antipsychotics, Anticholinergic effect, Mouse cerebral cortex

β-Adrenoceptors are subclassified into 3 subtypes (β1-β3). Among these, β3-adrenoceptors are present in various types of smooth muscle and are believed to play a role in relaxation responses of these muscles. β3-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 β3-adrenoceptors to produce relaxation responses in UBSM. In this study, to determine whether noradrenaline is a ligand of UBSM β3-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 β1-, β2-, and β3-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 β1-adrenoceptor antagonist), ICI-118,551 (3 × 10-8 M; selective β2-adrenoceptor antagonist), propranolol (10-7 M; non-selective β-adrenoceptor antagonist), and bupranolol (10-7 M; non-selective β-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 β3-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 β3-adrenoceptors to produce relaxation responses of UBSM in rats.

In yeast, mitochondria are passed on to daughter cells via the actin cable, motor protein Myo2, and adaptor protein Mmr1. They are released from the actin-myosin machinery after reaching the daughter cells. We report that Mmr1 is rapidly degraded by the ubiquitin-proteasome system in Saccharomyces cerevisiae. Redundant ubiquitin ligases Dma1 and Dma2 are responsible for Mmr1 ubiquitination. Dma1/2-mediated Mmr1 ubiquitination requires phosphorylation, most likely at S414 residue by Ste20 and Cla4. These kinases are mostly localized to the growing bud and nearly absent from mother cells, ensuring phosphorylation and ubiquitination of Mmr1 after the mitochondria enter the growing bud. In dma1Δ dma2Δ cells, transported mitochondria are first stacked at the bud-tip and then pulled back to the bud-neck. Stacked mitochondria in dma1Δ dma2Δ cells exhibit abnormal morphology, elevated respiratory activity, and increased level of reactive oxygen species, along with hypersensitivity to oxidative stresses. Collectively, spatiotemporally-regulated Mmr1 turnover guarantees mitochondrial homeostasis.

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 CaChanges 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.U46619, prostaglandin (PG) EProstanoids 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.

Docosahexaenoic acid (DHA, an n-3 polyunsaturated fatty acid) inhibits U46619 (a TP receptor agonist)- and prostaglandin F

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.

Yeast cells adapt to alkaline conditions by activating the Rim101 alkali-responsive pathway. Rim21 acts as a sensor in the Rim101 pathway and detects extracellular alkalization. Interestingly, Rim21 is also known to be activated by alterations involving the lipid asymmetry of the plasma membrane. In this study, we briefly summarize the mechanism of activation and the signal transduction cascade of the Rim101 pathway and propose a hypothesis on how Rim21 is able to detect distinct signals, particularly external alkalization, and altered lipid asymmetry. We found that external alkalization can suppress transbilayer movements of phospholipids between the two leaflets of the plasma membrane, which may lead to the disturbance of the lipid asymmetry of the plasma membrane. Therefore, we propose that external alteration is at least partly sensed by Rim21 through alterations in lipid asymmetry. Understanding this activation mechanism could greatly contribute to drug development against fungal infections.

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.

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.

The β-adrenoceptor (β-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 (β2-AR dominant), even in the presence of clorgiline, a monoamine oxidase-A inhibitor. In mouse trachea (β1-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 β3-AR partial agonist) in the presence of 3,5-dinitrocatechol plus propranolol. Our findings indicate that 1) MA may possess β2-AR agonistic action; 2) NMA and MA augment β2-AR-mediated tracheal relaxation in the absence of CA metabolic inhibitors, though themselves possessing β1-, β2-AR antagonistic action (β2 > β1); 3) DHPG exhibits β1-, β2-, β3-AR antagonistic action, and this is particularly marked for β3-AR. Our observations may help explain some of the pathologies associated with pheochromocytoma, which is characterized by increased CA metabolite levels.

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 Ca2+ 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) E2, PGF2α, and PGA2 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 Ca2+-free solution. U46619-enhanced basal tone was completely suppressed by verapamil, an inhibitor of voltage-dependent Ca2+ 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 Ca2+ channels (ROCCs)/store-operated Ca2+ 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.

Object We identified the β-adrenoceptor (β-AR) subtypes responsible for the relaxant responses to adrenaline (AD) and noradrenaline (NA) in the rat thoracic aorta and examined the role of cAMP which is involved in these relaxant responses. Methods The effects of β-AR antagonists or the adenylyl cyclase inhibitor SQ 22,536 on AD- and NA-induced relaxant responses in phenylephrine-induced contraction and increases in cAMP levels were examined in isolated, endothelium-denuded rat thoracic aorta segments. Results AD-induced relaxation was completely suppressed by propranolol (10−7 M) or by ICI-118,551 (10−8 M) plus atenolol (10−6 M), and was also very strongly inhibited by ICI-118,551 (10−8 M) alone. AD (10−5 M) increased tissue cAMP levels by approximately 1.9-fold compared with that in non-stimulated aortic tissue, but did not significantly increase cAMP levels in the presence of ICI-118,551 (10−8 M) or SQ 22,536 (10−4 M). AD-induced relaxation was strongly suppressed by SQ 22,536 (10−4 M). NA-induced relaxation was almost completely suppressed by atenolol (10−6 M) plus ICI-118,551 (10−8 M) although it was hardly affected by ICI-118,551 (10−8 M) alone. NA (10−5 M) increased tissue cAMP levels by approximately 2.2-fold compared with that in non-stimulated aortic tissue, but did not significantly increase cAMP levels in the presence of atenolol (10−6 M) or SQ 22,536 (10−4 M). NA-induced relaxation was strongly suppressed by SQ 22,536 (10−4 M). Conclusion In rat thoracic aorta, AD- and NA-induced relaxations, which are both strongly dependent on increased tissue cAMP levels, are mainly mediated through β2- and β1-adrenoceptors respectively.

Docosahexaenoic acid (DHA, an n-3 polyunsaturated fatty acid) inhibits U46619 (a TP receptor agonist)- and prostaglandin F2α-induced contractions in rat aorta and mesenteric arteries. However, whether these effects could be replicated in vasospasm-prone vessels, such as coronary and cerebral arteries, remains unknown. Here, we evaluated the changes in pig coronary and basilar artery tensions and intracellular Ca2+ concentrations in human prostanoid TP or FP receptor-expressing cells. We aimed to clarify whether DHA inhibits U46619- and prostaglandin F2α-induced contractions in spasm-prone blood vessels and determine if the TP receptor is the primary target for DHA. In both pig coronary and basilar arteries, DHA suppressed U46619- and prostaglandin F2α-induced sustained contractions in a concentration-dependent manner, but did not affect contractions induced by 80 mM KCl. SQ 29,548 (a TP receptor antagonist) suppressed U46619- and prostaglandin F2α-induced contractions by approximately 100% and 60%, respectively. DHA suppressed both U46619- and prostaglandin F2α-induced increases in intracellular Ca2+ concentrations in human TP receptor-expressing cells. However, DHA did not affect prostaglandin F2α-induced increases in intracellular Ca2+ concentrations in human FP receptor-expressing cells. These findings suggest that DHA potently inhibits TP receptor-mediated contractions in pig coronary and basilar arteries, and the primary mechanism underlying its inhibitory effects on arterial contractions involves inhibiting TP receptors.

Eph receptor tyrosine kinases are important for cancer development and progression as well as in cellular repulsive responses. We determined that SOCS box-containing protein SPSB4 destabilizes EphB2 cytoplasmic fragments. SPSB4 is a novel ubiquitin ligase regulating EphB2-dependent cell repulsive responses., Eph receptor tyrosine kinases and their ephrin ligands are overexpressed in various human cancers, including colorectal malignancies, suggesting important roles in many aspects of cancer development and progression as well as in cellular repulsive responses. The ectodomain of EphB2 receptor is cleaved by metalloproteinases (MMPs) MMP-2/MMP-9 and released into the extracellular space after stimulation by its ligand. The remaining membrane-associated fragment is further cleaved by the presenilin-dependent γ-secretase and releases an intracellular peptide that has tyrosine kinase activity. Although the cytoplasmic fragment is degraded by the proteasome, the responsible ubiquitin ligase has not been identified. Here, we show that SOCS box-containing protein SPSB4 polyubiquitinates EphB2 cytoplasmic fragment and that SPSB4 knockdown stabilizes the cytoplasmic fragment. Importantly, SPSB4 down-regulation enhances cell repulsive responses mediated by EphB2 stimulation. Altogether, we propose that SPSB4 is a previously unidentified ubiquitin ligase regulating EphB2-dependent cell repulsive responses.

We investigated the global landscape of Ca2+homeostasis in budding yeast based on high-dimensional chemical-genetic interaction profiles. The morphological responses of 62 Ca2+-sensitive (cls) mutants were quantitatively analyzed with the image processing program CalMorph after exposure to a high concentration of Ca2+. After a generalized linear model was applied, an analysis of covariance model was used to detect significant Ca2+–cls interactions. We found that high-dimensional, morphological Ca2+–cls interactions were mixed with positive (86%) and negative (14%) chemical-genetic interactions, whereas one-dimensional fitness Ca2+–cls interactions were all negative in principle. Clustering analysis with the interaction profiles revealed nine distinct gene groups, six of which were functionally associated. In addition, characterization of Ca2+–cls interactions revealed that morphology-based negative interactions are unique signatures of sensitized cellular processes and pathways. Principal component analysis was used to discriminate between suppression and enhancement of the Ca2+-sensitive phenotypes triggered by inactivation of calcineurin, a Ca2+-dependent phosphatase. Finally, similarity of the interaction profiles was used to reveal a connected network among the Ca2+homeostasis units acting in different cellular compartments. Our analyses of high-dimensional chemical-genetic interaction profiles provide novel insights into the intracellular network of yeast Ca2+homeostasis.

The polytopic plasma membrane protein Rim21 senses both the elevation of ambient pH and alterations in plasma membrane lipid asymmetry in the Rim101 pathway in budding yeast. Rim21 is known to undergo N-glycosylation, but the site and function of the glycosylation modification is not known. Using a systematic mutation analysis, we found that Rim21 is N-glycosylated at an unconventional motif located in the N-terminal extracellular region. The Rim21 mutant protein that failed to receive N-glycosylation showed prolonged protein lifetime compared to that of WT Rim21 protein. Although both the WT and mutant Rim21 localized to the plasma membrane, they exhibited different biochemical fractionation profiles. The mutant Rim21, but not WT Rim21, was mainly fractionated into the heavy membrane fraction. Further, compared to WT Rim21, mutant Rim21 was more easily solubilized with digitonin but was conversely more resistant to solubilization with Triton X-100. Despite these different biochemical properties from WT Rim21, mutant Rim21 protein could still activate the Rim101 pathway in response to external alkalization. Collectively, N-glycosylation of Rim21 is not indispensable for its activity as a sensor protein, but modulates the residence of Rim21 protein to some microdomains within the plasma membrane with distinct lipid conditions, thereby affecting its turnover.Key words: plasma membrane, lipid asymmetry, N-linked glycosylation, microdomain, Saccharomyces cerevisiae.

Aims β-Adrenoceptors (β-ADRs) mediating the relaxation of rat superior mesenteric arteries (SMAs) were pharmacologically identified, and the effects of chemical sympathetic denervation on β-ADR-mediated relaxation were examined. Main methods The tension changes of endothelium-denuded SMAs were isometrically recorded and the mRNA of endothelium-denuded SMA β-ADR was detected using RT-PCR. Key findings In endothelium-denuded SMAs contracted with ≥10−7 M phenylephrine (an α1-ADR agonist), isoprenaline (a β-ADR agonist)-induced relaxation was competitively inhibited by 3 × 10−9–10−8 M propranolol (a β1,2-ADR antagonist), but not further affected by ≥10−8 M propranolol. Although isoprenaline-induced relaxation was not affected by ICI-118,551 (10−9–10−8 M; a β2-ADR antagonist), it was competitively inhibited by atenolol (10−7–3 × 10−7 M; a β1-ADR antagonist) in the presence of ICI-118,551. In the presence of 10−7 M propranolol, isoprenaline- and CGP-12177A (a β3-ADR partial agonist)-induced relaxation was competitively inhibited by high concentrations of bupranolol (a β1,2,3-ADR antagonist), with pA2 values of 6.49 and 5.76, respectively. We detected the mRNA of β1- and β3-ADRs in endothelium-denuded SMAs. Treatment with 6-hydroxydopamine (a catecholaminergic neurotoxin) reduced maximal isoprenaline-induced relaxation in the presence and absence of 10−7 M propranolol, but not CGP-12177A-induced relaxation. Significance Isoprenaline-induced relaxation of rat SMAs is mediated by β1- and β3-ADRs. β-ADR-mediated relaxation of rat SMAs is shown to be attenuated by chemical sympathetic denervation. The differences in the effects of bupranolol and chemical sympathetic denervation on the responses to isoprenaline and CGP-12177A in rat SMAs might be explained by the possible presence of multiple β3-ADRs with different pharmacological properties.

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

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.

Angiotensin II (Ang II) is an intrinsic peptide having strong vasopressor effects, and thus, it plays an important role in the physiological regulation of blood pressure. The vasopressor effects of Ang II include direct contraction of myocardium and vascular smooth muscles (SMs) along with aldosterone-mediated sodium retention. In addition, indirect vascular contractions induced by noradrenaline (NA), the release of which is mediated through Ang II receptor type 1 (AT1) existing at the sympathetic nerve terminals (SNTs), also contribute to the vasopressor effects of Ang II. Stimulation of NA release from SNTs by Ang II also occurs in the myocardium leading to an increase in heart rate and cardiac contraction. Furthermore, Ang II enhances the contractions of non-vascular SMs, such as vas deferens, through induction of NA release from the SNTs. We have found that Ang II attenuated vagus nerve stimulation-induced bradycardia in a losartan-sensitive manner. This suggests that Ang II attenuates vagus nerve stimulation-induced bradycardia by inhibiting acetylcholine (ACh) release from the parasympathetic nerve terminals (PNTs) through activation of the AT1 receptor. Ang II was also reported to attenuate the release of ACh from the PNTs in SMs, such as stomach and airway, thus suppressing their contractile functions. There are, however, conflicting reports of the effects of Ang II on parasympathetic nerve-mediated contractile regulation of SMs. In this review, we have highlighted the relevant research articles including our experimental reports on the regulation of sympathetic and parasympathetic nerve-mediated excitation and contraction by Ang II along with the future prospects.