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α1-Adrenoceptors are involved in numerous physiological functions, including micturition. However, the pharmacological profile of the α1-adrenoceptor subtypes remains controversial. Here, we review the literature regarding α1-adrenoceptors in the lower urinary tract from the standpoint of α1L phenotype pharmacology. Among three α1-adrenoceptor subtypes (α1A, α1B and α1D), α1a-adrenoceptor mRNA is the most abundantly transcribed in the prostate, urethra and bladder neck of many species, including humans. In prostate homogenates or membrane preparations, α1A-adrenoceptors with high affinity for prazosin have been detected as radioligand binding sites. Functional α1-adrenoceptors in the prostate, urethra and bladder neck have low affinity for prazosin, suggesting the presence of an atypical α1-adrenoceptor phenotype (designated as α1L). The α1L-adrenoceptor occurs as a distinct binding entity from the α1A-adrenoceptor in intact segments of variety of tissues including prostate. Both the α1L- and α1A-adrenoceptors are specifically absent from Adra1A (α1a) gene-knockout mice. Transfection of α1a-adrenoceptor cDNA predominantly expresses α1A-phenotype in several cultured cell lines. However, in CHO cells, such transfection expresses α1L- and α1A-phenotypes. Under intact cell conditions, the α1L-phenotype is predominant when co-expressed with the receptor interacting protein, CRELD1α. In summary, recent pharmacological studies reveal that two distinct α1-adrenoceptor phenotypes (α1A and α1L) originate from a single Adra1A (α1a-adrenoceptor) gene, but adrenergic contractions in the lower urinary tract are predominantly mediated via the α1L-adrenoceptor. From the standpoint of phenotype pharmacology, it is likely that phenotype-based subtypes such as the α1L-adrenoceptor will become new targets for drug development and pharmacotherapy. LINKED ARTICLE This article is commented on by Ventura, pp. 1223-1225 of this issue. To view this commentary visit [ABSTRACT FROM AUTHOR]

Purpose: Sensory nerve terminals are highly distributed in the cornea, and regulate ocular surface sensation and homeostasis in response to various endogenous and exogenous stimuli. However, little is known about mediators regulating the physiological and pathophysiological activities of corneal sensory nerves. The aim of this study was to investigate the presence of cholinergic regulation in sensory nerves in the cornea., Methods: Localization of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (vAChT) was evaluated using western blotting and immunohistochemical analysis. The synthesis and liberation of acetylcholine from the cornea were assessed using corneal segments pre-incubated with [ 3 H]choline. The responsiveness of corneal neurons and nerves to cholinergic drugs was explored using calcium imaging with primary cultures of trigeminal ganglion neurons and extracellular recording from corneal preparations in guinea pigs., Results: ChAT, but not vAChT, was highly distributed in the corneal epithelium. In corneal segments, [ 3 H] acetylcholine was synthesized from [ 3 H]choline, and was also released in response to electrical stimuli. In cultured corneal neurons, the population sensitive to a transient receptor potential melastatin 8 (TRPM8) agonist exhibited high probability of responding to nicotine in a calcium imaging experiment. The firing frequency of cold-sensitive corneal nerves was increased by the application of nicotine, but diminished by an α4 nicotinic acetylcholine receptor antagonist., Conclusions: The corneal epithelium can synthesize and release acetylcholine. Corneal acetylcholine can excite sensory nerves via nicotinic receptors containing the α4 subunit. Therefore, corneal acetylcholine may be one of the important regulators of corneal nerve activity arranging ocular surface condition and sensation., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

How is the quantal size in neurotransmitter release adjusted for various firing levels? We explored the possible mechanisms that regulate acetylcholine (ACh) release from cholinergic interneurons using an ultra-mini superfusion system. After preloading [ 3 H]ACh in rat striatal cholinergic interneurons, the release was elicited by electrical stimulation under a condition in which presynaptic cholinergic and dopaminergic feedback was inhibited. [ 3 H]ACh release was reproducible at intervals of more than 10 min; shorter intervals resulted in reduced levels of ACh release. Upon persistent stimulation for 10 min, ACh release transiently increased, before gradually decreasing. Vesamicol, an inhibitor of the vesicular ACh transporter (VAChT), had no effect on the release induced by the first single pulse, but it reduced the release caused by subsequent pulses. Vesamicol also reduced the [ 3 H]ACh release evoked by multiple pulses, and the inhibition was enhanced by repetitive stimulation. The decreasing phase of [ 3 H]ACh release during persistent stimulation was accelerated by vesamicol treatment. Thus, it is likely that releasable ACh was slowly compensated for via VAChT during and after stimulation, changing the vesicular ACh content. In addition, ACh release per pulse decreased under high-frequency stimulation. The present results suggest that ACh release from striatal cholinergic interneurons may be adjusted by changes in the quantal size due to slow replenishment via VAChT, and by a reduction in release probability upon high-frequency stimulation. These two distinct processes likely enable the fine tuning of neurotransmission and neuroprotection/limitation against excessive output and have important physiological roles in the brain., (© 2023 International Society for Neurochemistry.)

Inflammatory bowel disease (IBD), which includes Crohn's disease and ulcerative colitis, is an intestinal disorder that causes prolonged inflammation of the gastrointestinal tract. Currently, the etiology of IBD is not fully understood and treatments are insufficient to completely cure the disease. In addition to absorbing essential nutrients, intestinal epithelial cells prevent the entry of foreign antigens (micro-organisms and undigested food) through mucus secretion and epithelial barrier formation. Disruption of the intestinal epithelial homeostasis exacerbates inflammation. Thus, the maintenance and reinforcement of epithelial function may have therapeutic benefits in the treatment of IBD. Muscarinic acetylcholine receptors (mAChRs) are G protein-coupled receptors for acetylcholine that are expressed in intestinal epithelial cells. Recent studies have revealed the role of mAChRs in the maintenance of intestinal epithelial homeostasis. The importance of non-neuronal acetylcholine in mAChR activation in epithelial cells has also been recognized. This review aimed to summarize recent advances in research on mAChRs for intestinal epithelial homeostasis and the involvement of non-neuronal acetylcholine systems, and highlight their potential as targets for IBD therapy.

It was reported that nicotinic acetylcholine receptor (nAChR)-mediated signaling pathways affect the proliferation and differentiation of pluripotent stem cells. However, detail expression profiles of nAChR genes were unrevealed in these cells. In this study, we comprehensively investigated the gene expression of α subunit of nAChRs (Chrna) during differentiation and induction of pluripotent stem cells. Mouse embryonic stem (ES) cells expressed multiple Chrna genes (Chrna3-5, 7 and 9) in undifferentiated status. Among them, Chrna9 was markedly down-regulated upon the differentiation into mesenchymal cell lineage. In mouse tissues and cells, Chrna9 was mainly expressed in testes, ES cells and embryonal F9 teratocarcinoma stem cells. Expression of Chrna9 gene was acutely reduced during differentiation of ES and F9 cells within 24 h. In contrast, Chrna9 expression was increased in induced pluripotent stem cells established from mouse embryonic fibroblast. It was shown by the reporter assays that T element-like sequence in the promoter region of Chrna9 gene is important for its activities in ES cells. Chrna9 was markedly reduced by siRNA-mediated knockdown of Tbx3, a pluripotency-related transcription factor of the T-box gene family. These results indicate that Chrna9 is a nAChR gene that are transcriptionally regulated by Tbx3 in undifferentiated pluripotent cells., (© 2023. The Author(s).)

Recent clinical studies indicate that dry eye is closely associated with psychiatric disorders such as depression and anxiety. Here, we investigated whether two types of mouse dry eye models showed depressive-like behavior in forced swim and sucrose preference tests, and whether voluntary wheel-running helped ameliorate depressive states. To reproduce the dry eye models, the exorbital lacrimal glands (ELG) or exorbital and intraorbital lacrimal glands (ELG+ILG) were bilaterally excised from male C57BL/6J mice. Tear volume was persistently reduced in both models, but the ELG+ILG excision mice exhibited more severe corneal damage than the ELG excision mice. In the forced swim and sucrose preference tests, the gland excision mice showed longer immobility and shorter climbing times, and lower sucrose preference than sham-operated mice, respectively, which appeared earlier in the ELG+ILG excision mice. Wheel-running activities were significantly lower in the ELG+ILG excision mice, but not in the ELG excision mice. After short-period wheel-running, the longer immobility times and the shorter climbing times in the forced swim completely disappeared in both models. Our results suggest that dry eyes might directly cause a depressive disorder that depends on the severity and duration of the ocular surface damage, and that voluntary motor activity could help recovery from a depressive state induced by dry eye., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Nakano, Nakazawa, He, Uwada, Kiyoi, Ishibashi and Masuoka.)

Cholinergic transmission underlies higher brain functions such as cognition and movement. To elucidate the process whereby acetylcholine (ACh) release is maintained and regulated in the central nervous system, uptake of [ 3 H]choline and subsequent synthesis and release of [ 3 H]ACh were investigated in rat striatal segments. Incubation with [ 3 H]choline elicited efficient uptake via high-affinity choline transporter-1, resulting in accumulation of [ 3 H]choline and [ 3 H]ACh. However, following inhibition of ACh esterase (AChE), incubation with [ 3 H]choline led predominantly to the accumulation of [ 3 H]ACh. Electrical stimulation and KCl depolarization selectively released [ 3 H]ACh but not [ 3 H]choline. [ 3 H]ACh release gradually declined upon repetitive stimulation, whereas the release was reproducible under inhibition of AChE. [ 3 H]ACh release was abolished after treatment with vesamicol, an inhibitor of vesicular ACh transporter. These results suggest that releasable ACh is continually replenished from the cytosol to releasable pools of cholinergic vesicles to maintain cholinergic transmission. [ 3 H]ACh release evoked by electrical stimulation was abolished by tetrodotoxin, but that induced by KCl was largely resistant. ACh release was Ca 2+ dependent and exhibited slightly different sensitivities to N- and P-type Ca 2+ channel toxins (ω-conotoxin GVIA and ω-agatoxin IVA, respectively) between both stimuli. [ 3 H]ACh release was negatively regulated by M2 muscarinic and D2 dopaminergic receptors. The present results suggest that inhibition of AChE within cholinergic neurons and of presynaptic negative regulation of ACh release contributes to maintenance and facilitation of cholinergic transmission, providing a potentially useful clue for the development of therapies for cholinergic dysfunction-associated disorders, in addition to inhibition of synaptic cleft AChE., (© 2021 International Society for Neurochemistry.)

17β-hydroxysteroid dehydrogenase type 3 (HSD17B3) converts androstenedione (A4) into testosterone (T), which regulates sex steroid production. Because various mutations of the HSD17B3 gene cause disorder of sex differentiation (DSD) in multiple mammalian species, it is very important to reveal the molecular characteristics of this gene in various species. Here, we revealed the open reading frame of the ovine HSD17B3 gene. Enzymatic activities of ovine HSD17B3 and HSD17B1 for converting A4 to T were detected using ovine androgen receptor-mediated transactivation in reporter assays. Although HSD17B3 also converted estrone to estradiol, this activity was much weaker than those of HSD17B1. Although ovine HSD17B3 has an amino acid sequence that is conserved compared with other mammalian species, it possesses two amino acid substitutions that are consistent with the reported variants of human HSD17B3. Substitutions of these amino acids in ovine HSD17B3 for those in human did not affect the enzymatic activities. However, enzymatic activities declined upon missense mutations of the HSD17B3 gene associated with 46,XY DSD, affecting amino acids that are conserved between these two species. The present study provides basic information and tools to investigate the molecular mechanisms behind DSD not only in ovine, but also in various mammalian species.

Aims: Chemoresistance remains a persistent challenge in advanced prostate cancer therapy. Probenecid reportedly inhibits multiple drug-efflux transporters; hence, it can be employed as a potential sensitizer for chemotherapy. In the present study, we evaluated the effects of probenecid on three-dimensional (3D)-cultures of prostate cancer cells., Main Methods: Prostate cancer cell lines, 22Rv1 and PC-3 were cultured as multicellular tumor spheroids. The effects of probenecid were evaluated using the MTT assay for viability, microscopy for spheroid size, and soft agar colony formation assay for anchorage-independent growth., Key Findings: The 3D-cultured 22Rv1 cells were less sensitive to cisplatin and doxorubicin than two-dimensional (2D) cell culture. Co-administration of probenecid at a low (100 or 300 μM), but not high (500 μM), concentration increased the sensitivity to cisplatin or doxorubicin in 22Rv1 spheroids. Probenecid increased the expression of ABCG2, a multidrug resistance transporter, in a dose-dependent manner. Furthermore, treatment with probenecid alone reduced the growth of 22Rv1 spheroids. Conversely, probenecid inhibited spheroid compaction rather than growth inhibition in 3D-cultured PC-3 cells. Moreover, probenecid inhibited colony formation of 22Rv1 and PC-3 cells in soft agar, as well as downregulated focal adhesion kinase (FAK), a crucial factor in anchorage-independent growth., Significance: In 3D-cultured prostate cancer cells, probenecid demonstrated pleiotropic effects such as chemosensitization, growth suppression, inhibition of spheroid compaction, and suppression of anchorage-independent growth. Elucidating the detailed mechanism underlying these probenecid actions could result in the identification of novel therapeutic targets toward the advanced prostate cancer., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Porcine steroid hormone profiles have some unique characteristics. We previously studied human and murine steroidogenesis using steroidogenic cells-derived from mesenchymal stem cells (MSCs). To investigate porcine steroidogenesis, we induced steroidogenic cells from porcine subcutaneous preadipocytes (PSPA cells), which originate from MSCs. Using cAMP, adenovirus-mediated introduction of steroidogenic factor-1 (SF-1)/adrenal 4-binding protein (Ad4BP) induced the differentiation of PSPA cells into sex steroid-producing cells. Introducing SF-1/Ad4BP also induced the aldo-keto reductase 1C1 (AKR1C1) gene. Porcine AKR1C1 had 17β-hydroxysteroid dehydrogenase activity, which converts androstenedione and 11-ketoandrostenedione into testosterone (T) and 11-ketotestosteorne (11KT). Furthermore, differentiated cells expressed hydroxysteroid 11β-dehydrogenase 2 (HSD11B2) and produced 11KT. HSD11B2 was expressed in testicular Leydig cells and the adrenal cortex. 11KT was present in the plasma of both immature male and female pigs, with slightly higher levels in the male pigs. T levels were much higher in the male pigs. It is noteworthy that in the female pigs, the 11KT levels were >10-fold higher than the T levels. However, castration altered the 11KT and T plasma profiles in the male pigs to near those of the females. 11KT induced endothelial nitric oxide synthase (eNOS) in porcine vascular endothelial cells. These results indicate that 11KT is produced in porcine adrenal glands and testes, and may regulate cardiovascular functions through eNOS expression., (Copyright © 2021. Published by Elsevier Ltd.)

Although 11-ketotestosterone (11KT) and testosterone (T) are major androgens in both teleosts and humans, their 5α-reduced derivatives produced by steroid 5α-reductase (SRD5A/srd5a), i.e., 11-ketodihydrotestosterone (11KDHT) and 5α-dihydrotestosterone (DHT), remains poorly characterized, especially in teleosts. In this study, we compared the presence and production of DHT and 11KDHT in Japanese eels and humans. Plasma 11KT concentrations were similar in both male and female eels, whereas T levels were much higher in females. In accordance with the levels of their precursors, 11KDHT levels did not show sexual dimorphism, whereas DHT levels were much higher in females. It is noteworthy that plasma DHT levels in female eels were higher than those in men. In addition, plasma 11KDHT was undetectable in both sexes in humans, despite the presence of 11KT. Three srd5a genes ( srd5a1 , srd5a2a and srd5a2b ) were cloned from eel gonads. All three srd5a genes were expressed in the ovary, whereas only both srd5a2 genes were expressed in the testis. Human SRD5A1 was expressed in testis, ovary and adrenal, whereas SRD5A2 was expressed only in testis. Human SRD5A1, SRD5A2 and both eel srd5a2 isoforms catalyzed the conversion of T and 11KT into DHT and 11KDHT, respectively, whereas only eel srd5a1 converted T into DHT. DHT and 11KDHT activated eel androgen receptor (ar)α-mediated transactivation as similar fashion to T and 11KT. In contrast, human AR and eel arβ were activated by DHT and11KDHT more strongly than T and 11KT. These results indicate that in teleosts, DHT and 11KDHT may be important 5α-reduced androgens produced in the gonads. In contrast, DHT is the only major 5α-reduced androgens in healthy humans., Competing Interests: Author YM was employed by the company ASKA Pharmaceutical Medical Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Yazawa, Inaba, Imamichi, Sekiguchi, Uwada, Islam, Orisaka, Mikami, Ida, Sato, Miyashiro, Takahashi, Khan, Suzuki, Umezawa and Kitano.)

PNU-120596 is a classical positive allosteric modulator (PAM) of α7 nicotinic acetylcholine receptor (α7 nAChR) and widely used to investigate the effect of α7 nAChR activation on several inflammation-associated diseases including rheumatoid arthritis, inflammatory bowel disease and cerebral ischemia. In this study, we report that PNU-120596 directly inhibits p38 mitogen-activated protein kinase (MAPK) activity. In 293A cells, p38 MAPK phosphorylation by several factors (oxidative stress, osmotic stress, TNF-α, or muscarinic stimulation) was inhibited by PNU-120596 as well as p38 MAPK inhibitor BIRB-796. Inhibition of p38 MAPK phosphorylation by PNU-120596 was not affected by α7 nAChR antagonist, methyllycaconitine (MLA). In vitro kinase assay revealed that PNU-120596 directly inhibits p38α MAPK-induced activating transcription factor 2 (ATF2) phosphorylation. MKK6-induced phosphorylation of p38α MAPK was also inhibited by PNU-120596. Real-time monitoring of binding to p38α MAPK using fluoroprobe SKF-86002 showed quite rapid binding of PNU-120596 compared to BIRB-796 which is known as a slow binder. Finally, we showed that PNU-120596 suppressed LPS-induced phosphorylation of p38 MAPK and expression of inflammatory factors including TNF-α, IL-6 and COX-2, independent on α7 nAChR activity in microglial cell BV-2. Thus, PNU-120596 might exert an anti-inflammatory effect through not only α7 nAChR potentiation but also direct inhibition of p38 MAPK., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Short-chain fatty acids (SCFAs), including acetate, butyrate, and propionate, are produced when colonic bacteria in the human gastrointestinal tract ferment undigested fibers. Free fatty acid receptor 2 (FFA2) and FFA3 are G-protein-coupled receptors recently identified as SCFA receptors that may modulate inflammation. We previously showed through in vitro experiments that SCFAs activate FFA2 and FFA3, thereby mitigating inflammation in human renal cortical epithelial cells. This study used a murine model of adenine-induced renal failure to investigate whether or not SCFAs can prevent the progression of renal damage. We also examined whether or not these FFA2 and FFA3 proteins have some roles in this protective mechanism in vivo. Immunohistochemical analyses of mouse kidneys showed that FFA2 and FFA3 proteins were expressed mainly in the distal renal tubules and collecting tubules. First, we observed that the administration of propionate mitigated the renal dysfunction and pathological deterioration caused by adenine. Consistent with this, the expression of inflammatory cytokines and fibrosis-related genes was reduced. Furthermore, the mitigation of adenine-induced renal damage by the administration of propionate was significantly attenuated in FFA2 -/- and FFA3 -/- mice. Therefore, the administration of propionate significantly protects against adenine-induced renal failure, at least in part, via the FFA2 and FFA3 pathways. Our data suggest that FFA2 and FFA3 are potential new therapeutic targets for preventing or delaying the progression of chronic kidney disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Hepatocellular carcinoma (HCC) is a major cause of cancer death worldwide and establishment of new chemotherapies for HCC is urgently needed. GPR41 [free fatty acid receptor 3 (FFA3)] is a G protein-coupled receptor for short chain fatty acids, including acetate, propionate, and butyrate. In our previous study, we showed that propionate enhances the cytotoxic effect of cisplatin in HCC cells and that this mechanism is dependent on inhibition of histone deacetylases (HDACs) via GPR41/FFA3. However, the antitumor action of GPR41/FFA3 has not been elucidated., Methods: In this study, we examined AR420626 as a GPR41-selective agonist in HepG2 and HLE cells. Nude mice were used for HepG2 xenograft studies. The apoptotic effect of AR420626 was evaluated using flow cytometry analysis. Expression of apoptosis-related proteins and HDACs was evaluated by Western immunoblot. Gene silencing of HDAC 3/5/7 and GPR41 was performed using small interfering RNA. Expression of TNF-α mRNA was evaluated by TaqMan real-time polymerase chain reaction., Results: We found that AR420626, a selective GPR41/FFA3 agonist, suppressed growth of HepG2 xenografts and inhibited proliferation of HCC cells by inducing apoptosis. AR420626 induced proteasome activation through mTOR phosphorylation, which reduced HDAC proteins, and then increased expression of TNF-α., Conclusion: AR420626, a selective GPR41/FFA3 agonist, may be a candidate as a therapeutic agent for HCC., Competing Interests: Conflict of interest statement: The authors declare that there is no conflict of interest., (© The Author(s), 2020.)

17β-Hydroxysteroid dehydrogenases (17β-HSDs) catalyze the reduction of 17-ketosteroids and the oxidation of 17β-hydroxysteroids to regulate the production of androgens and estrogens. Among them, 17β-HSD type 3 (HSD17B3) is expressed almost exclusively in testicular Leydig cells and contributes to development of male sexual characteristics by converting androstenedione (A4) to testosterone (T). Mutations of HSD17B3 genes cause a 46,XY disorder of sexual development (46,XY DSD) as a result of low T production. Therefore, the evaluation of 17β-HSD3 enzymatic activity is important for understanding and diagnosing this disorder. We adapted a method that easily evaluates enzymatic activity of 17β-HSD3 by quantifying the conversion from A4 to T using androgen receptor (AR)-mediated transactivation. HEK293 cells were transduced to express human HSD17B3, and incubated medium containing A4. Depending on the incubation time with HSD17B3-expressing cells, the culture media progressively increased luciferase activities in CV-1 cells, transfected with the AR expression vector and androgen-responsive reporter. Culture medium from HSD17B1 and HSD17B5-expressing cells also increased the luciferase activities. This system is also applicable to detect the conversion of 11-ketoandrostenedione to 11-ketotestosterone by HSD17B3. Establishment of HEK293 cells expressing various missense mutations in the HSD17B3 gene associated with 46,XY DSD revealed that this system is effective to evaluate the enzymatic activities of mutant proteins., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Ketone bodies, including acetoacetate and β-hydroxybutyrate (βOHB), are produced from acetyl coenzyme A in the liver and then secreted into the blood. These molecules are a source of energy for peripheral tissues during exercise or fasting. βOHB has been reported to inhibit histone deacetylases (HDACs) 1, 3, and 4 in human embryonic kidney 293 cells. Thus, βOHB may regulate epigenetics by modulating HDACs. There have been several reports that the administration of βOHB or induction of a physiological state of ketosis has an antitumor effect; however, the mechanism remains unclear. The aim of this study was to investigate whether βOHB enhances cisplatin-induced apoptosis in hepatocellular carcinoma (HCC) cells by modulating activity and/or expression of HDACs. We found that βOHB significantly enhanced cisplatin-induced apoptosis and cleavage of caspase-3 and -8 in HCC cells. Further, βOHB significantly decreased the expression of HDCA 3/5/6 and survivin in liver hepatocellular (HepG2) cells. In HDAC3/6 gene silencing, survivin expression was significantly decreased, and cisplatin-induced cleavage of caspase-3 was significantly enhanced compared with control in HepG2 cells. In conclusion, βOHB enhanced cisplatin-induced apoptosis via HDAC3/6 inhibition/survivin axis in HepG2 cells, which suggests that βOHB could be a new adjuvant agent for cisplatin chemotherapy., (Copyright © 2019. Production and hosting by Elsevier B.V.)

Calcium influx via store-operated calcium entry (SOCE) has an important role for regulation of vast majority of cellular physiological events. MAPK signalling is also another pivotal modulator of many cellular functions. However, the relationship between SOCE and MAPK is not well understood. In this study, we elucidated the involvement of SOCE in Gα q/11 protein-mediated activation of p38 MAPK in an intestinal epithelial cell line HT-29/B6. In this cell line, we previously showed that the stimulation of M3 muscarinic acetylcholine receptor (M3-mAChR) but not histamine H1 receptor (H1R) led to phosphorylation of p38 MAPK which suppressed tumor necrosis factor-α (TNF-α)-induced NF-κB signalling through ADAM17 protease-mediated shedding of TNF receptor-1 (TNFR1). First, we found that stimulation of M3-mAChR and protease-activated receptor-2 (PAR-2) but not H1R induced persistent upregulation of cytosolic Ca 2+ concentration through SOCE. Activation of M3-mAChR or PAR-2 also suppressed TNF-α-induced NF-κB phosphorylation, which was dependent on the p38 MAPK activity. Time course experiments revealed that M3-mAChR stimulation evoked intracellular Ca 2+ -dependent early phase p38 MAPK phosphorylation and extracellular Ca 2+ -dependent later phase p38 MAPK phosphorylation. This later phase p38 MAPK phosphorylation, evoked by M3-mAChRs or PAR-2, was abolished by inhibition of SOCE. Thapsigargin or ionomycin also phosphorylate p38 MAPK by Ca 2+ influx through SOCE, leading to suppression of TNF-α-induced NF-κB phosphorylation. Finally, we showed that p38 MAPK was essential for thapsigargin-induced cleavage of TNFR1 and suppression of TNF-α-induced NF-κB phosphorylation. In conclusion, SOCE is important for p38 MAPK phosphorylation and is involved in TNF-α signalling suppression., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Cyclooxygenase 2 (COX-2) is an inducible rate-limiting enzyme for prostanoid production. Because COX-2 represents one of the inducible genes in mouse mesenchymal stem cells upon differentiation into Leydig cells, we investigated COX-2 expression and production of prostaglandin (PG) in Leydig cells. Although COX-2 was undetectable in mouse testis, it was transiently induced in Leydig cells by human chorionic gonadotropin (hCG) administration. Consistent with the finding that Leydig cells expressed aldo-keto reductase 1B7 (PGF synthase) and PGE synthase 2, induction of COX-2 by hCG caused a marked increase in testicular PGF 2α and PGE 2 levels. Using mouse Leydig cell tumor-derived MA-10 cells as a model, it was indicated by reporter assays and electron mobility shift assays that transcription of the COX-2 gene was activated by CCAAT/enhancer-binding protein β (C/EBPβ) with cAMP-stimulation. C/EBPβ expression was induced by cAMP-stimulation, whereas expression of C/EBP homolog protein (CHOP) was robustly downregulated. Transfection of CHOP expression plasmid inhibited cAMP-induced COX-2 promoter activity. In addition, CHOP reduced constitutive COX-2 expression in other mouse Leydig cell tumor-derived TM3 cells. These results indicate that COX-2 is induced in Leydig cells by activation of C/EBPβ via reduction of CHOP expression upon gonadotropin-stimulation to produce PGF 2α and PGE 2 ., (© 2019 Wiley Periodicals, Inc.)

Regulation of neurotransmitter release in the central nervous system is complex. Here, we investigated regulatory mechanisms for acetylcholine (ACh) release from cholinergic neurons by performing superfusion experiments with rat striatal segments after labelling the cellular ACh pool with [ 3 H]choline. Electrical stimulation-evoked pronounced [ 3 H]ACh release from cholinergic neurons. The estimated quantity of [ 3 H]ACh release per pulse of electrical stimulation was reduced by an increase in stimulus frequency, showing an inverse correlation between release probability of ACh and neuronal excitation. ACh release was also negatively regulated by pre-synaptic muscarinic ACh receptors (mAChRs). The autoinhibition induced by released ACh was predominantly suppressed by the M 2 -selective antagonist AF-DX 116, partially inhibited by M 3 -selective darifenacin, and minimally by M 4 -selective PD 102807. Other subtype-selective antagonists had no effect at subtype-selective concentrations. ACh esterase (AChE) inhibitors (diisopropylfluorophosphate, donepezil and galantamine) at concentrations that mostly inhibit esterase activity reduced [ 3 H]ACh release, and the reduction was abolished by treatment with atropine. This implies that pre-synaptic autoreceptors are activated more after blockade of ACh hydrolysis, leading to autoinhibition of ACh release and consequent reduction in synaptic ACh concentrations. [ 3 H]efflux was also enhanced by ACh uptake inhibitors (100 μM hemicholinium-3 and physostigmine), regardless of ACh hydrolysis. This study shows that synaptic ACh concentrations in striatal cholinergic neurons are regulated in a complex manner by many factors such as release probability, pre-synaptic M 2 /M 3 /M 4 mAChRs, AChE and post-synaptic ACh uptake, and provides important information about cholinergic neurotransmission for future exploration of therapeutic strategies for Alzheimer's and other central nervous system diseases. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/openscience-badges/., (© 2019 International Society for Neurochemistry.)

Hippocampal cholinergic activity enhances long-term potentiation (LTP) of synaptic transmission in intrahippocampal circuits and regulates cognitive function. We recently demonstrated intracellular distribution of functional M1-muscarinic acetylcholine receptors (mAChRs) and neuronal uptake of acetylcholine (ACh) in the central nervous system. Here we examined whether endogenous ACh acts on intracellular M1-mAChRs following its uptake and causes cholinergic facilitation of hippocampal LTP. ACh esterase (AChE) activities and [ 3 H]ACh uptake was measured in rat hippocampal segments. LTP of evoked field excitatory postsynaptic potentials at CA1 synapses was induced by high frequency stimulation in hippocampal slices. Pretreatment with diisopropylfluorophosphate (DFP) irreversibly inhibited AChE, augmented ACh uptake, and significantly enhanced the LTP. This cholinergic facilitation was inhibited by pirenzepine, a membrane-permeable M1 antagonist, while only the early stage of cholinergic facilitation was inhibited by a membrane-impermeable M1 antagonist, muscarinic toxin 7. Tetraethylammonium (TEA) inhibited ACh uptake in hippocampal segments and selectively suppressed late stage cholinergic facilitation without changing the early stage. In contrast, LTP in DFP-untreated slices was not affected by the muscarinic antagonists and TEA. Carbachol (CCh; an AChE-resistant muscarinic agonist) competed with ACh for its uptake and produced cholinergic facilitation of LTP in DFP-untreated slices. The late stage of CCh-induced facilitation was also selectively inhibited by TEA. Our results suggest that when AChE is inactivated by inhibitors, LTP in hippocampal slices is significantly enhanced by endogenous ACh and that cholinergic facilitation is caused by direct activation of cell-surface M1-mAChRs and subsequent activation of intracellular M1-mAChRs after ACh uptake., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)

Ovaries represent one of the primary steroidogenic organs, producing estrogen and progesterone under the regulation of gonadotropins during the estrous cycle. Gonadotropins fluctuate the expression of various steroidogenesis-related genes, such as those encoding steroidogenic enzymes, cholesterol deliverer, and electronic transporter. Steroidogenic factor-1 (SF-1)/adrenal 4-binding protein (Ad4BP)/NR5A1 and liver receptor homolog-1 (LRH-1) play important roles in these phenomena via transcriptional regulation. With the aid of cAMP, SF-1/Ad4BP and LRH-1 can induce the differentiation of stem cells into steroidogenic cells. This model is a useful tool for studying the molecular mechanisms of steroidogenesis. In this article, we will provide insight into the transcriptional regulation of steroidogenesis-related genes in ovaries that are revealed from stem cell-derived steroidogenic cells. Using the cells derived from the model, novel SF-1/Ad4BP- and LRH-1-regulated genes were identified by combined DNA microarray and promoter tiling array analyses. The interaction of SF-1/Ad4BP and LRH-1 with transcriptional regulators in the regulation of ovarian steroidogenesis was also revealed.

Aims: β-Hydroxybutyrate (βOHB) is a metabolic intermediate that constitutes about 70% of ketone bodies produced in liver from oxidation of fatty acids released from adipose tissue. A recent study showed that βOHB inhibits HDAC1, 3 and 4 (classes I and IIa) in human embryonic kidney 293 (HEK293) cells. Therefore, βOHB could regulate epigenetics via modulating HDACs. However, little is known about the protective effect of βOHB on renal cells through epigenetics. The aim of this study is to investigate whether βOHB reduces cisplatin-induced nephrotoxicity in human renal cortical epithelial (HRCE) cells by modulating HDACs., Main Methods: In this study, we used human renal cortical epithelial (HRCE) cells. The anti-apoptotic effect of βOHB was evaluated using flow cytometry analysis. The expression of apoptosis-related proteins and HDACs was evaluated by western immunoblot., Key Findings: The results showed that βOHB significantly reduced cisplatin-induced apoptosis in HRCE cells. Furthermore, βOHB significantly reduced cisplatin-induced cleavage of caspase-3, acetylation of histone H3, and phosphorylation of AMP-activated kinase. This anti-apoptotic effect of βOHB was markedly attenuated by an inhibitor of HDAC4/5, and βOHB-mediated suppression of cleavage of caspase3 was significantly blocked by siRNA-induced gene silencing of HDAC5., Significance: βOHB attenuates cisplatin-induced apoptosis by activation of HDAC5 in HRCE cells, suggesting that βOHB may be a new therapeutic agent for cisplatin nephropathy., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate are generated by microbial fermentation of indigestible fiber by gut flora. SCFAs are ligands of two orphan G protein-coupled receptors, GPR41 and GPR43, that modulate cell proliferation and induce apoptosis. However, it is unclear if SCFAs enhance the effects of chemotherapy in a GPR41- or GPR43-dependent manner. The aim of this study was to investigate whether SCFAs, and particularly propionate, activate GPR41 or GPR43, and thereby enhance the antitumor effects of cisplatin in HepG2 human hepatocellular carcinoma (HCC) cells. The inhibitory effects of propionate and cisplatin on proliferation of HCC cells were determined by MTS assay. Changes in apoptosis rate were analyzed by flow cytometry. The effects of combined propionate and cisplatin on these properties in HCC cells were significantly higher than those of cisplatin alone. With combined treatment, the levels of cleaved caspase-3, active caspase-3 forms, and acetylated histone H3 were enhanced in a GPR41-dependent manner; expression of histone deacetylases (HDAC) 3, 4, 5, 6, 8 proteins was significantly reduced; and induction of TNF-α expression was significantly enhanced. These results suggest that propionate and cisplatin synergistically and significantly induce apoptosis of HepG2 cells by increasing expression of autocrine TNF-α via reduction of HDACs through GPR41 signaling. From clinical and translational perspectives, our data suggest that a combination of propionate with cisplatin may have better therapeutic effects on HCC compared with conventional treatment, and that a selective GPR41 agonist may be a candidate as an adjuvant therapeutic agent for HCC., Competing Interests: CONFLICTS OF INTEREST The authors declare that there are no conflicts of interest.

Protein kinase Cγ (PKCγ) knock-out (KO) animals exhibit symptoms of Parkinson's disease (PD), including dopaminergic neuronal loss in the substantia nigra. However, the PKCγ substrates responsible for the survival of dopaminergic neurons in vivo have not yet been elucidated. Previously, we found 10 potent substrates in the striatum of PKCγ-KO mice. Here, we focused on cysteine string protein α (CSPα), a protein from the heat shock protein (HSP) 40 cochaperone families localized on synaptic vesicles. We found that in cultured cells, PKCγ phosphorylates CSPα at serine (Ser) 10 and Ser34. Additionally, apoptosis was found to have been enhanced by the overexpression of a phosphorylation-null mutant of CSPα, CSPα(S10A/S34A). Compared with wild-type (WT) CSPα, the CSPα(S10A/S34A) mutant had a weaker interaction with HSP70. However, in sharp contrast, a phosphomimetic CSPα(S10D/S34D) mutant, compared with WT CSPα, had a stronger interaction with HSP70. In addition, total levels of synaptosomal-associated protein (SNAP) 25, a main downstream target of the HSC70/HSP70 chaperone complex, were found to have decreased by the CSPα(S10A/S34A) mutant through increased ubiquitination of SNAP25 in PC12 cells. In the striatum of 2-year-old male PKCγ-KO mice, decreased phosphorylation levels of CSPα and decreased SNAP25 protein levels were observed. These findings indicate the phosphorylation of CSPα by PKCγ may protect the presynaptic terminal from neurodegeneration. The PKCγ-CSPα-HSC70/HSP70-SNAP25 axis, because of its role in protecting the presynaptic terminal, may provide a new therapeutic target for the treatment of PD. SIGNIFICANCE STATEMENT Cysteine string protein α (CSPα) is a protein belonging to the heat shock protein (HSP) 40 cochaperone families localized on synaptic vesicles, which maintain the presynaptic terminal. However, the function of CSPα phosphorylation by protein kinase C (PKC) for neuronal cell survival remains unclear. The experiments presented here demonstrate that PKCγ phosphorylates CSPα at serine (Ser) 10 and Ser34. CSPα phosphorylation at Ser10 and Ser34 by PKCγ protects the presynaptic terminal by promoting HSP70 chaperone activity. This report suggests that CSPα phosphorylation, because of its role in modulating HSP70 chaperone activity, may be a target for the treatment of neurodegeneration., (Copyright © 2018 the authors 0270-6474/18/380278-13$15.00/0.)

In addition to hydrolysis by acetylcholine esterase (AChE), acetylcholine (ACh) is also directly taken up into brain tissues. In this study, we examined whether the uptake of ACh is involved in the regulation of synaptic ACh concentrations. Superfusion experiments with rat striatal segments pre-incubated with [ 3 H]choline were performed using an ultra-mini superfusion vessel, which was developed to minimize superfusate retention within the vessel. Hemicholinium-3 (HC-3) at concentrations less than 1 μM, selectively inhibited the uptake of [ 3 H]choline by the high affinity-choline transporter 1 and had no effect on basal and electrically evoked [ 3 H]efflux in superfusion experiments. In contrast, HC-3 at higher concentrations, as well as tetraethylammonium (>10 μM), which inhibited the uptake of both [ 3 H]choline and [ 3 H]ACh, increased basal [ 3 H]overflow and potentiated electrically evoked [ 3 H]efflux. These effects of HC-3 and tetraethylammonium were also observed under conditions where tissue AChE was irreversibly inactivated by diisopropylfluorophosphate. Specifically, the potentiation of evoked [ 3 H]efflux was significantly higher in AChE-inactivated preparations and was attenuated by atropine. On the other hand, striatal segments pre-incubated with [ 3 H]ACh failed to increase [ 3 H]overflow in response to electrical stimulation. These results show that synaptic ACh concentrations are significantly regulated by the postsynaptic uptake of ACh, as well as by AChE hydrolysis and modulation of ACh release mediated through presynaptic muscarinic ACh receptors. In addition, these data suggest that the recycling of ACh-derived choline may be minor in cholinergic terminals. This study reveals a new mechanism of cholinergic transmission in the central nervous system., (© 2017 International Society for Neurochemistry.)

Diethylstilbestrol (DES), a strong estrogenic compound, is well-known to affect the reproductive system. In this study, we investigated the effects of DES administration on gonadotropin levels and ovarian steroidogenesis in prepubertal rats. DES treatment acutely reduced serum LH levels, followed by a reduction in the expression of various steroidogenesis-related genes in theca cells. Serum FSH levels were almost unaffected by DES-treatment, even though Cyp19a1 expression was markedly reduced. Serum progesterone, testosterone and estradiol levels were also declined at this time. LH levels recovered from 12 h after DES-treatment and gradually increased until 96 h with a reduction of ERα expression observed in the pituitary. Steroidogenesis-related genes were also up-regulated during this time, except for Cyp17a1 and Cyp19a1. Consistent with observed gene expression pattern, serum testosterone and estradiol concentrations were maintained at lower levels, even though progesterone levels recovered. DES-treatment induced the inducible nitric oxide synthase (iNOS) in granulosa cells, and a nitric oxide generator markedly repressed Cyp19a1 expression in cultured granulosa cells. These results indicate that DES inhibits thecal androgen production via suppression of pituitary LH secretion and ovarian Cyp17a1 expression. In addition, DES represses Cyp19a1 expression by inducing iNOS gene expression for continuous inhibition of estrogen production in granulosa cells.

Intestinal epithelial cells form a tight barrier to act as selective physical barriers, repelling hostile substances. Tumor necrosis factor-α (TNF-α) is a well characterized pro-inflammatory cytokine which can compromise intestinal barrier function and the suppression of TNF-α function is important for treatment of inflammatory bowel disease (IBD). In this study, we investigated the contribution of G-protein-coupled receptor (GPCR)-induced signalling pathways to the maintenance of epithelial barrier function. We first demonstrated the existence of functional muscarinic M3 and histamine H1 receptors in colonic epithelial cell HT-29/B6. As we previously reported, muscarinic M3 receptor prevented TNF-α-induced barrier disruption through acceleration of TNF receptor (TNFR) shedding which is carried out by TNF-α converting enzyme (TACE). M3 receptor-mediated suppression of TNF-α function depends on Gα q/11 protein, however, histamine H1 receptor could not ameliorate TNF-α function, while which could induce Gα q/11 dependent intracellular Ca 2+ mobilization. We found that p38 MAPK was predominantly phosphorylated by M3 receptor through Gα q/11 protein, whereas H1 receptor barely upregulated the phosphorylation. Inhibition of p38 MAPK abolished M3 receptor-mediated TNFR shedding and suppression of TNF-α-induced NF-κB signalling. The p38 MAPK was also involved in TACE- mediated EGFR transactivation followed by ERK1/2 phosphorylation. These results indicate that not H1 but M3 receptor-induced activation of p38 MAPK might contribute to the maintenance of epithelial barrier function through down-regulation of TNF-α signalling and activation of EGFR., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Steroid hormones are mainly produced in adrenal glands and gonads. Because steroid hormones play vital roles in various physiological processes, replacement of deficient steroid hormones by hormone replacement therapy (HRT) is necessary for patients with adrenal and gonadal failure. In addition to HRT, tissue regeneration using stem cells is predicted to provide novel therapy. Among various stem cell types, mesenchymal stem cells can be differentiated into steroidogenic cells following ectopic expression of nuclear receptor (NR) 5A subfamily proteins, steroidogenic factor-1 (also known as adrenal 4 binding protein) and liver receptor homolog-1, with the aid of cAMP signaling. Conversely, these approaches cannot be applied to pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells, because of poor survival following cytotoxic expression of NR5A subfamily proteins. However, if pluripotent stem cells are first differentiated through mesenchymal lineage, they can also be differentiated into steroidogenic cells via NR5A subfamily protein expression. This approach offers a potential suitable cells for future regenerative medicine and gene therapy for diseases caused by steroidogenesis deficiencies. It represents a powerful tool to investigate the molecular mechanisms involved in steroidogenesis. This article highlights our own and current research on the induction of steroidogenic cells from various stem cells. We also discuss the future direction of their clinical application.

Functional acetylcholine receptors (AChRs) were recently demonstrated to exist not only in the plasma membrane but also intracellularly in brain tissues. In order to activate intracellular AChRs, endogenous hydrophilic ACh must cross the plasma membrane. Here, we examined the pharmacological characteristics of this process, including whether it is mediated by active ACh uptake. When ACh esterase (AChE) was suppressed by diisopropylfluorophosphate, [ 3 H]ACh was effectively taken up into segments of rat cerebral cortex and other brain regions, in contrast to peripheral tissues such as liver and kidney. The uptake of [ 3 H]ACh in rat cerebral cortex was temperature-dependent, and the uptake capacity was comparable to that of [ 3 H]choline. However, [ 3 H]ACh uptake was inhibited by lower concentrations of ACh, carbachol, tetraethylammonium (TEA), compared with uptake of [ 3 H]choline. Uptake of [ 3 H]ACh was also inhibited by several organic cations, including choline, hemicholinium-3 (HC-3), quinidine, decynium 22, clonidine, diphenhydramine, but was little affected by some amino acids and biogenic amines, corticosterone, spermine, atropine, and tetrodotoxin. Unlike diisopropylfluorophosphate, several ACh esterase inhibitors, including drugs for Alzheimer's disease, such as donepezil, galantamine, and rivastigmine, also suppressed the uptake of [ 3 H]ACh, but not [ 3 H]choline. These results indicate that in the brain, ACh is specifically taken up through a unique transport system with different pharmacological properties from known organic cation transporters (OCTs), and suggest that this mechanism may be involved in intracellular cholinergic transmission in the brain., (© 2016 International Society for Neurochemistry.)

Impaired intestinal barrier function is one of the critical issues in inflammatory bowel diseases. The aim of this study is to investigate muscarinic cholinoceptor (mAChR)-mediated signaling for the amelioration of cytokine-induced barrier dysfunction in intestinal epithelium. Rat colon challenged with TNF-α and interferon γ reduced transepithelial electrical resistance (TER). This barrier injury was attenuated by muscarinic stimulation. In HT-29/B6 intestinal epithelial cells, muscarinic stimulation suppressed TNF-α-induced activation of NF-κB signaling and barrier disruption. Finally, muscarinic stimulation promoted the shedding of TNFR1, which would be a mechanism for the attenuation of TNF-α/NF-κB signaling and barrier disruption via mAChR., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Steroidogenic factor-1 (SF-1) and liver receptor homolog-1 (LRH-1) belong to the nuclear receptor superfamily and are categorized as orphan receptors. In addition to other nuclear receptors, these play roles in various physiological phenomena by regulating the transcription of target genes. Both factors share very similar structures and exhibit common functions. Of these, the roles of SF-1 and LRH-1 in steroidogenesis are the most important, especially that of SF-1, which was originally discovered and named to reflect such roles. SF-1 and LRH-1 are essential for steroid hormone production in gonads and adrenal glands through the regulation of various steroidogenesis-related genes. As SF-1 is also necessary for the development of gonads and adrenal glands, it is also considered a master regulator of steroidogenesis. Recent studies have clearly demonstrated that LRH-1 also represents another master regulator of steroidogenesis, which similarly to SF-1, can induce differentiation of non-steroidogenic stem cells into steroidogenic cells. Here, we review the functions of both factors in these steroidogenesis-related phenomena.

Regulation of intestinal secretion is important for body fluid homeostasis. We investigated the role of three MAP kinases (MAPKs) as negative regulators in muscarinic cholinoceptor (mAChR)-mediated intestinal secretion in mice. Electrophysiological analyses revealed that mAChR stimulation enhanced intestinal chloride secretion, which was further augmented by the inhibition of JNK but not by that of ERK or p38 with specific inhibitors SP600125, U0126 or SB203580, respectively. Immunoblot analyses in colonic mucosa showed that mAChR stimulation increased MAPKs phosphorylation that was suppressed by the specific inhibitor for each MAPK. This suggests that JNK is a major negative regulator in mAChR-induced intestinal secretion., (Copyright © 2014 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.)

Background and Purpose: The pharmacological properties of particular receptors have recently been suggested to vary under different conditions. We compared the pharmacological properties of the α1B -adrenoceptor subtype in various tissue preparations and under various conditions., Experimental Approach: [(3) H]-prazosin binding to α1B -adrenoceptors in rat liver (segments, dispersed hepatocytes and homogenates) was assessed and the pharmacological profiles were compared with the functional and binding profiles in rat carotid artery and recombinant α1B -adrenoceptors., Key Results: In association and saturation-binding experiments with rat liver, binding affinity for [(3) H]-prazosin varied significantly between preparations (KD value approximately ten times higher in segments than in homogenates). The binding profile for various drugs in liver segments also deviated from the representative α1B -adrenoceptor profile observed in liver homogenates and recombinant receptors. L-765,314 and ALS-77, selective antagonists of α1B -adrenoceptors, showed high binding and antagonist affinities in liver homogenates and recombinant α1B -adrenoceptors. However, binding affinities for both ligands in the segments of rat liver and carotid artery were 10 times lower, and the antagonist potencies in α1B -adrenoceptor-mediated contractions of carotid artery were more than 100 times lower than the representative α1B -adrenoceptor profile., Conclusions and Implications: In contrast to the consistent profile of recombinant α1B -adrenoceptors, the pharmacological profile of native α1B -adrenoceptors of rat liver and carotid artery varied markedly under various receptor environments, showing significantly different binding properties between intact tissues and homogenates, and dissociation between functional and binding affinities. In addition to conventional 'subtype' characterization, 'phenotype' pharmacology must be considered in native receptor evaluations in vivo and in future pharmacotherapy., (© 2014 The British Pharmacological Society.)

The M1 muscarinic acetylcholine receptor (M1-mAChR, encoded by CHRM1) is a G-protein-coupled membrane receptor that is activated by extracellular cholinergic stimuli. Recent investigations have revealed the intracellular localization of M1-mAChR. In this study, we observed constitutive internalization of M1-mAChR in mouse neuroblastoma N1E-115 cells without agonist stimulation. Constitutive internalization depended on dynamin, clathrin and the adaptor protein-2 (AP-2) complex. A WxxI motif in the M1-mAChR C-terminus is essential for its constitutive internalization, given that replacement of W(442) or I(445) with alanine residues abolished constitutive internalization. This WxxI motif resembles YxxΦ, which is the canonical binding motif for the μ2 subunit of the AP-2 complex. The M1-mAChR C-terminal WxxI motif interacted with AP-2 μ2. W442A and I445A mutants of the M1-mAChR C-terminal sequence lost AP-2-μ2-binding activity, whereas the W442Y mutant bound more effectively than wild type. Consistent with these results, W442A and I445A M1-mAChR mutants selectively localized to the cell surface. By contrast, the W442Y receptor mutant was found only at intracellular sites. Our data indicate that the cellular distribution of M1-mAChR is governed by the C-terminal tryptophan-based motif, which mediates constitutive internalization., (© 2014. Published by The Company of Biologists Ltd.)

Impairment of epithelial barrier is observed in various intestinal disorders including inflammatory bowel diseases (IBD). Numerous factors may cause temporary damage of the intestinal epithelium. A complex network of highly divergent factors regulates healing of the epithelium to prevent inflammatory response. However, the exact repair mechanisms involved in maintaining homeostatic intestinal barrier integrity remain to be clarified. In this study, we demonstrate that activation of M1 muscarinic acetylcholine receptor (mAChR) augments the restitution of epithelial barrier function in T84 cell monolayers after ethanol-induced epithelial injury, via ERK-dependent phosphorylation of focal adhesion kinase (FAK). We have shown that ethanol injury decreased the transepithelial electrical resistance (TER) along with the reduction of ERK and FAK phosphorylation. Carbachol (CCh) increased ERK and FAK phosphorylation with enhanced TER recovery, which was completely blocked by either MT-7 (M1 antagonist) or atropine. The CCh-induced enhancement of TER recovery was also blocked by either U0126 (ERK pathway inhibitor) or PF-228 (FAK inhibitor). Treatment of T84 cell monolayers with interferon-γ (IFN-γ) impaired the barrier function with the reduction of FAK phosphorylation. The CCh-induced ERK and FAK phosphorylation were also attenuated by the IFN-γ treatment. Immunological and binding experiments exhibited a significant reduction of M1 mAChR after IFN-γ treatment. The reduction of M1 mAChR in inflammatory area was also observed in surgical specimens from IBD patients, using immunohistochemical analysis. These findings provide important clues regarding mechanisms by which M1 mAChR participates in the maintenance of intestinal barrier function under not only physiological but also pathological conditions., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background and Purpose: Two distinct α1 -adrenoceptor phenotypes (α1A and α1L ) have recently been demonstrated to originate from a single α1A -adrenoceptor gene. Here, we examined the agonist profiles of recombinant α1A and α1L phenotypes and of lower urinary tract (LUT) α1 -adrenoceptors., Experimental Approach: A series of drugs (A61603, Ro 115-1240, NS-49 , MK017 and ESR1150) originally developed for stress urinary incontinence (SUI) therapy were used to stimulate recombinant α1A - and α1L -adrenoceptor phenotypes, and their potencies and intrinsic activity estimated from Ca(2+) responses. Agonist-induced contractions were also examined in LUT tissues of rats and humans and in human mesenteric artery and rat tail artery., Key Results: All the drugs were potent agonists of the α1A -adrenoceptor compared with the α1L -adrenoceptor phenotype. Among them, Ro 115-1240 was shown to be an α1A -specific partial agonist that produced partial contractions through α1A -adrenoceptors in rat prostate and tail artery, but not in the other LUT tissues and human mesenteric artery. In contrast, P-come 102 showed full agonist activity at α1A - and α1L -adrenoceptors, but was less selective than noradrenaline for α1A -adrenoceptors. Like noradrenaline, P-come 102 was highly potent at inducing contractions in all of the LUT tissues tested. However, the potency and intrinsic activity of P-come 102 were significantly lower than those of noradrenaline in human mesenteric artery., Conclusions and Implications: The α1A - and α1L -adrenoceptor phenotypes and LUT α1 -adrenoceptors were demonstrated to have distinct agonist profiles. As adrenergic contractions in LUT are predominantly mediated through α1L -adrenoceptors, the development of α1L -selective agonists may provide clinically useful drugs for SUI therapy., (© 2013 The British Pharmacological Society.)

Muscarinic acetylcholine receptors (mAChRs) are well known to transmit extracellular cholinergic signals into the cytoplasm from their position on the cell surface. However, we show here that M1-mAChRs are also highly expressed on intracellular membranes in neurons of the telencephalon and activate signaling cascades distinct from those of cell surface receptors, contributing uniquely to synaptic plasticity. Radioligand-binding experiments with cell-permeable and -impermeable ligands and immunohistochemical observations revealed intracellular and surface distributions of M1-mAChRs in the hippocampus and cortex of rats, mice, and humans, in contrast to the selective occurrence on the cell surface in other tissues. All intracellular muscarinic-binding sites were abolished in M1-mAChR-gene-knockout mice. Activation of cell surface M1-mAChRs in rat hippocampal neurons evoked phosphatidylinositol hydrolysis and network oscillations at theta rhythm, and transiently enhanced long-term potentiation. On the other hand, activation of intracellular M1-mAChRs phosphorylated extracellular-regulated kinase 1/2 and gradually enhanced long-term potentiation. Our data thus demonstrate that M1-mAChRs function at both surface and intracellular sites in telencephalon neurons including the hippocampus, suggesting a new mode of cholinergic transmission in the central nervous system., (© 2013 International Society for Neurochemistry.)

Background: Muscarinic acetylcholine receptors (mAChRs) are major regulators of gut epithelial functions. However, the precise subtype composition has not been clarified., Methods: We characterized the pharmacological profile of mAChRs on mouse colonic crypts, employing [(3)H]-N-methyl scopolamine chloride as a radioligand and several subtype-selective chemicals, and the functional aspect by measuring short-circuit current (I sc) in Ussing chambers and by evaluating MAP kinase phosphorylation in mouse colonic mucosal sheets., Results: The mAChRs were detected on the crypts (K d = 163.2 ± 32.3 pM, B max = 47.3 ± 2.6 fmol/mg of total cell protein). Muscarinic toxin 7 (MT-7, M1 subtype selective) gave a displacement curve with high affinity, but there was a part insensitive to MT-7 (18.8 ± 0.4 % of the total specific binding). The MT-7-insensitive component was displaced completely by darifenacin (M3 selective) with high affinity. ACh induced an increase in I sc, which was significantly enhanced by MT-7 but was completely inhibited by darifenacin or atropine. Colitis induction resulted in a significant decrease in the density of mAChRs, which occurred mainly in the MT-7-sensitive component (M1 subtype). Immunological experiments exhibited a reduction of M1 but not of M3 signal after colitis induction. Muscarinic stimulation induced an increase in MAP kinase phosphorylation, which was completely suppressed by MT-7 and was attenuated by inflammation, in mouse colonic epithelium., Conclusions: These results suggest that mAChRs in mouse colonic epithelial cells consist of two subtypes, M1 (80 %) and M3 (20 %). The major M1 subtype was likely to regulate epithelial chloride secretion negatively and was susceptible to inflammation and may be relevant to inflammatory gut dysfunction.

Aims: Recombinant systems have been used for evaluating the properties of G-protein-coupled receptors (GPCRs) on the assumption of cell surface expression. However, many GPCRs, including muscarinic acetylcholine receptors (mAChRs), have also been reported to be distributed in intracellular organelles in native tissues and cell lines. In this study, we compared the pharmacological profiles of exogenously and endogenously expressed M1-mAChRs, and evaluated the functional properties of these receptors., Main Methods: Recombinant M1-mAChRs were expressed exogenously in Chinese hamster ovary cells (CHO-M1 cells) and compared with endogenously expressed M1-mAChRs in N1E-115 neuroblastoma cells. The pharmacological and functional profiles were evaluated using cell-permeable antagonists (1-quinuclidinyl-benzilate (QNB), pirenzepine and atropine) and cell-impermeable antagonists (N-methylscopolamine (NMS) or MT-7)., Key Findings: M1-mAChRs were seen at the cell surface and intracellular sites in both cell lines. Under whole cell conditions, intracellular M1-mAChRs were mainly recognized by cell-permeable ligands, but scarcely by cell-impermeable ligands (at less than 100nM). In CHO-M1 cells, M1-mAChR activation by carbachol resulted in Ca(2+) mobilization, ERK1/2 phosphorylation and a reduction in thymidine incorporation, all of which were completely inhibited by MT-7, indicating the involvement of surface M1-mAChRs. In N1E-115 cells, Ca(2+) mobilization occurred through surface M1-mAChRs, whereas ERK1/2 phosphorylation and acceleration of thymidine incorporation were mediated through intracellular M1-mAChRs., Significance: Exogenous and endogenous M1-mAChRs are present at both the cell surface and the intracellular organelles, and the pharmacological properties of geographically distinct M1-mAChRs are different, and may depend on cell background and/or exogenous or endogenous origin., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Nicotinic acetylcholine receptors (nAChRs) of the cerebral cortex and cerebellum of rats were evaluated by a radioligand binding assay, employing tissue segments, or homogenates as materials. [(3)H]-epibatidine specifically bound to nAChRs in rat cortex or cerebellum, but the dissociation constants for [(3)H]-epibatidine differed between segments and homogenates (187 pM for segments and 42 pM for homogenates in the cortex and 160 pM for segments and 84 pM for homogenates in the cerebellum). The abundance of total nAChRs was approximately 310 fmol/mg protein in the segments of cortex and 170 fmol/mg protein in the segments of cerebellum, which were significantly higher than those estimated in the homogenates (115 fmol/mg protein in the homogenates of the cortex and 76 fmol/mg protein in the homogenates of the cerebellum). Most of the [(3)H]-epibatidine binding sites in the cortex segments (approximately 70% of the population) showed high affinity for nicotine (pK(i) = 7.9), dihydro-β-erythroidine, and cytisine, but the binding sites in the cerebellum segments had slightly lower affinity for nicotine (pK(i) = 7.1). An upregulation of nAChRs by chronic administration of nicotine was observed in the cortex segments but not in the cerebellum segments with [(3)H]-epibatidine as a ligand. The upregulation in the cortex was caused by a specific increase in the high-affinity sites for nicotine (probably α4β2). The present study shows that the native environment of nAChRs is important for a precise quantitative as well as qualitative estimation of nAChRs in rat brain.

Distinct pharmacological phenotypes of muscarinic acetylcholine receptors (mAChRs) have been proposed. We compared the pharmacological profiles of mAChRs in intact segments and homogenates of rat cerebral cortex and other tissues by using radioligand binding assays with [(3)H]N-methylscopolamine ([(3)H]NMS). Recombinant M(1) and M(3) mAChRs were also examined. The density of mAChRs detected by [(3)H]NMS binding to rat cerebral cortex segments and homogenates was the same (approximately 1400 fmol/mg tissue protein), but the dissociation constant of [(3)H]NMS was significantly different (1400-1700 pM in segments and 260 pM in homogenates). A wide variation in [(3)H]NMS binding affinity was also observed among the segments of other tissues (ranging from 139 pM in urinary bladder muscle to 1130 pM in the hippocampus). The mAChRs of cerebral cortex were composed of M(1), M(2), M(3), and M(4) subtypes, which showed typical subtype pharmacology in the homogenates. However, in the cortex segments the M(3) subtype showed a low selectivity for M(3) antagonists (darifenacin, solifenacin) and was not distinguished by the M(3) antagonists from the other subtypes. Recombinant M(1) and M(3) mAChRs showed high affinity for [(3)H]NMS and subtype-specific pharmacology for each tested ligand. The present binding study under conditions where tissue integrity was kept demonstrates a wide variation in [(3)H]NMS binding affinity among mAChRs of many rat tissues and the presence of an atypical M(3) phenotype in the cerebral cortex, suggesting that the pharmacological properties of mAChRs are not necessarily constant, rather they may be significantly modified by tissue integrity and tissue type.

Signaling by muscarinic agonists is thought to result from the activation of cell surface acetylcholine receptors (mAChRs) that transmit extracellular signals to intracellular systems. In N1E-115 neuroblastoma cells, we detected both plasma membrane and intracellular M(1) -mAChRs using both biochemical and pharmacological methods. In intact cells, both plasma membrane and intracellular M(1) -mAChRs were detected by the hydrophobic ligand probe, 1-quinuclidinyl-[phenyl-4-(3) H]-benzilate ([(3) H]-QNB) whereas the hydrophilic probe, 1-[N-methyl-(3) H] scopolamine ([(3) H]-NMS), detected only cell surface receptors. These probes detected comparable numbers of receptors in isolated membrane preparations. Immunohistochemical studies with M(1) -mAChR antibody also detected both cell-surface and intracellular M(1) -mAChRs. Carbachol-stimulated phosphatidylinositol hydrolysis and Ca(2+) mobilization were completely inhibited by a cell-impermeable M(1) antagonist, muscarinic toxin -7 and the G(q/11) inhibitor YM-254890. However, carbachol-stimulated extracellular-regulated kinase 1/2 activation was unaffected by muscarinic toxin-7, but was blocked by the cell-permeable antagonist, pirenzepine. extracellular regulated kinase 1/2 phosphorylation was resistant to blockade of G(q/11) (YM-254890) and protein kinase C (bisindolylmaleimide I). Our data suggest that the geographically distinct M(1) -mAChRs (cell surface versus intracellular) can signal via unique signaling pathways that are differentially sensitive to cell-impermeable versus cell-permeable antagonists. Our data are of potential physiological relevance to signaling that affects both cognitive and neurodegenerative processes., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

We found that the small ubiquitin-related modifier (SUMO) conjugation reaction can be visualized simply by incubating green fluorescent protein (GFP)-SUMO-1 with permeabilized cells in the presence of ATP for 15 min. Neither special equipment for protein purification nor highly developed skills for recombinant technologies is required, making the assay potentially applicable to large-scale drug-screening strategies for the identification of drugs that can inhibit or enhance SUMOylation.

The small ubiquitin-related modifier 2/3 (SUMO2/3) can be post-translationally conjugated to a wide variety of proteins constituting chromatin, the platform for genetic and epigenetic regulation. Nevertheless, it is unclear how SUMO2/3 and SUMO2/3-modified proteins are delivered to the chromatin fibers. Here we report that the largest subunit of chromatin assembly factor 1 (CAF-1), human p150, interacts directly and preferentially with SUMO2/3. Amino acid residue of 98-105 in p150 is essential and sufficient for SUMO2/3 interaction. p150-SUMO2/3 interaction coincided with regions that replicate chromatin fibers, because accumulation of the proliferating cell nuclear antigen (PCNA), and incorporation of bromodeoxyuridine (BrdU) were detected at foci co-localized with both p150 and SUMO2/3 during the S-phase in a cell line expressing epitope-tagged p150. Although inhibition of SUMO2/3 expression had only a small effect on p150 deposition on the replication sites, depletion of p150 led to delocalization of SUMO2/3 from the replication foci. Furthermore, p150 mutants deficient in SUMO2/3 interaction, caused a major reduction of SUMO2/3 at the replication foci. Thus, our findings suggest an expanded role of p150 as a SUMO2/3-interacting factor, and raise the intriguing possibility that p150 plays a role in promoting delivery of SUMO2/3 or SUMO2/3-modified proteins (or both) on chromatin fibers during replication., (Copyright 2009 Elsevier Inc. All rights reserved.)

We previously described the establishment of a binary vector system that allows co-expression of SUMO conjugation enzymes and a target protein of interest, leading to efficient SUMO modification and the production of a large amount of recombinant SUMO-modified proteins in Escherichia coli. The advantages of this E. coli expression/modification approach include scalability of experiments, low cost, fast growth, and a lack of proteases that cleave the isopeptide linkage between SUMO and the target protein. Thus, this E. coli method provides a useful alternative to authentic SUMO modification assays, such as in vitro SUMO conjugation and in vivo SUMO modification using baculovirus or mammalian cell culture, that are usually complicated, time-consuming and expensive.

Small ubiquitin-related modifiers, SUMO-2/3 and SUMO-1, are involved in gene regulation and nuclear structures. However, little is known about the roles of SUMO, in heterochromatin formation of mammalian cells. Here we demonstrate that SUMOs directly interact with human MCAF1, which forms complexes with either the methyl-CpG-binding protein MBD1 or SETDB1, which trimethylates histone H3 at lysine 9 (H3-K9) in the presence of MCAF1. Modification of MBD1 with either SUMO-2/3 or SUMO-1 facilitated the interaction between MBD1 and MCAF1, suggesting that SUMOylation links the methylation of DNA and histones. In a cultured human cell line, SUMOs were localized in MBD1- and MCAF1-containing heterochromatin regions that were enriched in trimethyl-H3-K9 and the heterochromatin proteins HP1beta and HP1gamma. Specific knockdown of either SUMO-2/3 or SUMO-1 induced dissociation of MCAF1, trimethyl-H3-K9, and the HP1 proteins from the MBD1-containing heterochromatin foci, suggesting a requirement for SUMOs for heterochromatin assembly. These findings provide insights into the roles of SUMOylation in the regulation of heterochromatin formation and gene silencing.