Induction of potato variants with enhanced resistance to common scab disease via cell culture is applicable to a cultivar developed in Japan, but the effect of using the phytotoxin thaxtomin A is restrictive.

To induce potato variants with enhanced resistance to common scab disease that retain the desirable agronomic traits of the original cultivars, we used a cell culture technique that employs thaxtomin A, the primary phytotoxin that induces scab symptoms. We induced 24 variants from the potato cultivar 'Saya-akane', developed in Japan, and selected two with enhanced resistance to the disease by growing them in planters with bacteriainoculated soil and in a field infested with the disease. We also examined toxin tolerance in micro-tubers of variants that showed a lower degree or percentage of infection in the glasshouse screening, and found no significant difference relative to the original cultivar. To clarify the effect of using thaxtomin A, we examined the efficiency of induction of the potential enhanced resistance by comparing the degree of infection among variants grown in planters with inoculated soil. We observed no significant difference between variants induced on culture medium with and without the toxin. These results suggest that the effect of using the toxin as a positive selection agent is restrictive and that most resistance-enhancing mutations are induced by the cell culture procedure itself.

Decreased Monoamine Oxidase A Expression and Activity in the Bladders of Rats With Partial Outlet Obstruction.

OBJECTIVE: To investigate changes in expression and activity of monoamine oxidase A (MAO-A) in rats with partial bladder outlet obstruction (pBOO). Previous studies suggested that monoamines, such as serotonin (5-hydroxytryptamine [5-HT]) and noradrenaline, are involved in bladder hyperactivity secondary to pBOO. However, little is known about the role of MAO-A, an enzyme oxidizing 5-hydroxytryptamine and noradrenalin, in the pathogenesis. MATERIALS AND METHODS: Female Sprague Dawley rats were subjected to sham or pBOO operations for 7 days, then their bladders were isolated. MAO-A protein levels in the bladder were examined by Western blotting. MAO-A activity was measured by the commercially available MAO-Glo Assay kit. In addition, MAO-A distribution in the bladder was examined by immunohistochemistry. RESULTS: Weights of the bladders from rats with pBOO were increased about 3.5-fold, compared with those from sham rats. Significant decreases in MAO-A protein and activity levels were observed in whole bladder of rats with pBOO compared with those of sham rats. By immunohistochemistry, it was firstly demonstrated that MAO-A was predominantly expressed in the detrusor layer of the sham rat bladders. The intensity of staining was decreased after pBOO operation. CONCLUSION: We demonstrated, for the first time, the distribution of MAO-A in the bladder and the pathologic changes in MAO-A protein and activity levels in rats with pBOO. This marked decrease in MAO-A potentially resulting in increased monoamine levels, especially in the detrusor of rat bladder, might be a key factor explaining the mechanism of bladder overactivity associated with pBOO.

Piezo1 expression increases in rat bladder after partial bladder outlet obstruction.

AIMS: For patients with benign prostatic hyperplasia (BPH), storage symptoms due to bladder dysfunction are bothersome, and that mechanism elucidation is needed. Piezo1, a mechanically activated ion channel, is believed to play a role in sensing bladder distension. To investigate the involvement of Piezo1 in bladder dysfunction, we examined the expression and distribution of Piezo1 and neurofilament (NF-L) to understand pathological alterations in rat bladders with partial bladder outlet obstruction (pBOO), an animal model of BPH. MAIN METHODS: Female Sprague-Dawley rats were subjected to sham or pBOO operations. On days 3, 7, and 14 after pBOO, Piezo1 mRNA levels in the bladder were examined by quantitative real-time PCR. The expression of light NF-L was also examined by western blotting. On day 7, the distributions of Piezo1 were examined by in situ hybridization. KEY FINDINGS: The expression levels of Piezo1 mRNA in whole bladder were significantly increased from days 3 to 14 after pBOO. On day 7 in pBOO rats, significant increases in Piezo1 mRNA were observed in the detrusor layer as well as the suburothelial layer, while the predominant distribution was observed in the urothelium of sham rats. Coinciding with the increase in Piezo1, the decreases in NF-L expression were observed in the bladder from pBOO rats. SIGNIFICANCE: The increase in Piezo1 in pBOO rat bladders might be involved in the compensatory mechanism associated with bladder denervation including the decrease in NF-L. Inhibition of Piezo-1 may be a new therapeutic approach to ameliorate the storage dysfunction shown in pBOO.

Mast Cell Accumulation and Degranulation in Rat Bladder with Partial Outlet Obstruction.

INTRODUCTION: Benign prostatic hyperplasia causes partial bladder outlet obstruction (pBOO), and many patients with pBOO are affected by not only voiding symptoms but also storage symptoms. We previously suggested that enhancement of 5-hydroxytryptamine (5-HT)-induced bladder contraction in the pBOO bladder may be one cause of storage symptoms. However, little is known about the presence of 5-HT in rat bladders. In this study, we hypothesized that mast cells are a source of 5-HT and investigated the distribution of mast cells and 5-HT in the bladders of rats with pBOO. METHODS: The bladders of female Sprague-Dawley rats were subjected to pBOO and sham operations for 1 week, were isolated, and were fixed for light or electron microscopy. Mast cells and 5-HT in the bladders were detected by toluidine blue staining and immunohistochemical staining, respectively. The mast cells were counted under a light microscope. Degranulated mast cells were observed under an electron microscope and counted under a light microscope. RESULTS: Mast cells were present in the mucosa/submucosa region in sham rat bladders. Their number was increased in the detrusor muscle/subserosa/serosa region, especially the subserosal layer, in pBOO rat bladders. The localization of mast cells almost matched that of 5-HT-positive cells in consecutive sections. Degranulated mast cells were present in sham and pBOO rat bladders, but the proportion of degranulated mast cells was significantly increased in every region in pBOO rat bladders compared with that in sham rat bladders. CONCLUSION: These results suggest that mast cells contain 5-HT and are more abundant locally in the subserosal layer of pBOO rat bladders. 5-HT released from mast cells could stimulate 5-HT2 receptors on the detrusor muscle, and this may underlie storage symptoms. FUNDING: Asahi Kasei Pharma Corp.

Increased expression of 5-HT(2A) and 5-HT(2B) receptors in detrusor muscle after partial bladder outlet obstruction in rats.

Serotonin (5-hydroxytryptamine; 5-HT)-induced bladder contraction is enhanced after partial bladder outlet obstruction (pBOO) in rats. We investigated time-dependent changes in bladder contraction and expression of 5-HT(2A) and 5-HT(2B) receptor mRNA in bladder tissue to elucidate the mechanism of this enhancement. On day 3 and 7 after pBOO, contractile responses of isolated rat bladder strips to 5-HT were increased compared with that in sham-operated rats; on day 14, the response had decreased to the same level as that in sham rat bladders. In contrast, carbacholinduced contraction was not enhanced by pBOO at any time point. In sham rats, 5-HT(2A) receptor mRNA was expressed in the urothelium, and 5-HT(2B) receptor mRNA was expressed in the detrusor muscle layer. In pBOO rats, both receptor mRNAs were increased in the detrusor muscle and subserosal layers, but not in the urothelium. The increase of 5-HT(2A) receptor mRNA was maintained from day 3 to day 14 after pBOO, and 5-HT(2B) receptor mRNA was increased on day 7 after pBOO. These results suggested that pBOO induced up-regulation of the 5-HT(2A) and 5-HT(2B) receptors in the detrusor muscle and subserosal layers of the bladder, and such up-regulation may be related to the enhanced bladder contractile response to 5-HT.

5-Hydroxytryptamine-induced bladder hyperactivity via the 5-HT2A receptor in partial bladder outlet obstruction in rats.

We investigated the effects of partial bladder outlet obstruction (BOO) on the function and gene expression of 5-hydroxytryptamine (5-HT) receptor subtypes in rat bladder. Isometric contractions of the isolated bladders from sham-operated control and BOO rats were examined. The contractile responses to 5-HT were significantly increased in BOO rat bladder strips, while the responses to KCl, carbachol, or phenylephrine were not different from the control. The 5-HT-induced hypercontraction in BOO rat bladder strips was inhibited by ketanserin, a 5-HT(2A) receptor antagonist. The contractile responses to 5-HT in bladder strips were not affected by urothelium removal from the intact bladder. The gene expression of 5-HT receptor subtypes in the bladders was analyzed by RT-PCR. The mRNA expression of the 5-HT(2A), 5-HT(2B), 5-HT(2C), 5-HT(4), and 5-HT(7) receptors was detected in both the control and BOO rat bladders. Quantitative RT-PCR analysis showed there was a significant increase of 5-HT(2A) receptor mRNA in the BOO rat bladder compared with the control bladder. On the other hand, the gene expression of the 5-HT(4) receptor was not changed in the BOO rat bladder. These results suggest that the increased contractile responses to 5-HT in BOO rat bladder may be partly caused by 5-HT(2A) receptor upregulation in the detrusor smooth muscles.

Naftopidil inhibits 5-hydroxytryptamine-induced bladder contraction in rats.

Naftopidil is an α(1D) and α(1A) subtype-selective α(1)-adrenoceptor antagonist that has been used to treat lower urinary tract symptoms of benign prostatic hyperplasia. In this study, we investigated the effects of naftopidil on 5-hydroxytryptamine (5-HT)-induced rat bladder contraction (10(-8)-10(-4) M). Naftopidil (0.3, 1, and 3 µM) inhibited 5-HT-induced bladder contraction in a concentration-dependent manner. On the other hand, other α(1)-adrenoceptor antagonists, tamsulosin, silodosin or prazosin, did not inhibit 5-HT-induced bladder contraction. The 5-HT-induced bladder contraction was inhibited by both ketanserin and 4-(4-fluoronaphthalen-1-yl)-6-propan-2-ylpyrimidin-2-amine (RS127445), serotonin 5-HT(2A) and 5-HT(2B) receptor antagonists, respectively. In addition, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and α-methyl-5-HT, 5-HT(2A) and 5-HT(2) receptor agonists, respectively, induced bladder contraction. The 5-HT-induced bladder contraction was not inhibited by N-[2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl]-N-pyridin-2-yl-cyclohexanecarboxamide (WAY-100635), [1-[2[(methylsulfonyl)amino]ethyl]-4-piperidinyl]methyl-1-methyl-1H-indole-3-carboxylate (GR113808) or (R)-3-[2-[2-(4-methylpiperidin-1-yl)ethyl]pyrrolidine-1-sulphonyl]phenol (SB269970), 5-HT(1A), 5-HT(4) and 5-HT(7) receptor antagonists, respectively. Naftopidil inhibited both the 5-HT(2A) and 5-HT(2) receptor agonists-induced bladder contractions. Naftopidil binds to the human 5-HT(2A) and 5-HT(2B) receptors with pKi values of 6.55 and 7.82, respectively. These results suggest that naftopidil inhibits 5-HT-induced bladder contraction via blockade of the 5-HT(2A) and 5-HT(2B) receptors in rats. Furthermore, 5-HT-induced bladder contraction was enhanced in bladder strips obtained from bladder outlet obstructed rats, with this contraction inhibited by naftopidil. The beneficial effects of naftopidil on storage symptoms such as urinary frequency and nocturia in patients with benign prostatic hyperplasia may be due, in part, to the blockade of the 5-HT(2A) and 5-HT(2B) receptors in the bladder.

Substitution effect, absorption, and fluorescence behaviors of 11,12-benzo-1,7,10,13-tetraoxa-4-aza- cyclopentadec-11-ene (benzoaza-15-crown-5) derivatives upon cation complexation in solvent extraction.

Substitution effect, absorption, and fluorescence behaviors of some benzoaza-15-crown-5 derivatives upon cation complexation in solvent extraction were studied. The introduction of a substituent on the nitrogen atom in benzoaza-15-crown-5 enhanced extractabilities in the solvent extraction of aqueous alkali metal picrates. The nondonating substituents raised the cation selectivity for Na(+) over K(+), but the donating substituents reduced the cation selectivity. The absorption and fluorescence spectral behavior was different with the alkali metal cations.