Changes in the expression of α-tocopherol-related genes in liver and mammary gland biopsy specimens of peripartum dairy cows.

Blood α-tocopherol (α-Toc) concentrations decline gradually throughout the prepartum period, reaching the nadir after calving in dairy cows. The 6 α-Toc-related molecules [α-Toc transfer protein (TTPA); afamin; scavenger receptor class B, Type I; ATP-binding cassette transporter A1; tocopherol-associated protein (SEC14L2); and cytochrome P450 family 4, subfamily F, polypeptide 2 (CYP4F2)] are expressed in liver and other peripheral tissues. These molecules could regulate α-Toc transport, blood concentrations, and metabolism of α-Toc. Therefore, the aim of this study was to evaluate the changes in the expression of α-Toc-related genes in liver and mammary gland tissues of dairy cows around calving, which have remained elusive until now. In experiment (Exp.) 1, 28 multiparous Holstein cows were used (from -5 to 6 wk relative to parturition) to monitor the changes in dietary α-Toc intake, blood concentrations of α-Toc, and lipoproteins; in Exp. 2, 7 peripartum Holstein cows were used (from -4 to 4 wk relative to parturition) for liver tissue biopsy; and in Exp. 3, 10 peripartum Holstein cows were used (from -8 to 6 wk relative to parturition) to carry out the mammary gland tissue biopsy and milk sampling. In Exp. 1, the serum α-Toc concentrations declined gradually with decreasing amount of α-Toc intake and plasma high-density lipoprotein concentrations toward calving time. However, in the early lactation period after calving, serum α-Toc concentrations remained at a lower concentration despite the recovery of α-Toc intake and plasma high-density lipoprotein concentrations. In Exp. 2, just after calving, the TTPA, SEC14L2, afamin, and albumin mRNA expression levels in the liver were temporarily downregulated, and the hepatic mRNA levels of endoplasmic reticulum stress-induced unfolded protein response markers and acute-phase response marker increased at calving. In Exp. 3, the concentrations of α-Toc in colostrum were greater than those in precolostrum (samples were collected at wk -1 relative to parturition) and mature milk. The expression of TTPA, SEC14L2, and CYP4F2 mRNA in bovine mammary gland tissue was detected. However, TTPA and SEC14L2 mRNA expressions showed the opposite trends: the expression levels of TTPA mRNA peaked whereas SEC14L2 mRNA reached a nadir at calving. These results indicate that the expression of α-Toc-related genes involved in specific α-Toc transfer and metabolism in the liver and mammary gland are altered during calving. Moreover, these changes might be associated with the maintenance of lower serum α-Toc concentrations after calving.

Roles of histone H3.5 in human spermatogenesis and spermatogenic disorders.

Histone H3.5 (H3.5) is a newly identified histone variant highly expressed in the human testis. We have reported the crystal structure, instability of the H3.5 nucleosome and accumulation around transcription start sites, mainly in primary spermatocytes, but its role in human spermatogenesis remains poorly understood. Testicular biopsy specimens from 30 men (mean age: 35 years) with non-obstructive azoospermia (NOA) who underwent microdissection testicular sperm extraction and 23 men with obstructive azoospermia (OA) were included. An H3.5-specific mouse monoclonal antibody recognizing an H3.5-specific synthetic peptide was generated, and immunohistological staining for H3.5 and proliferating cell nuclear antigen (PCNA) was performed on Bouin's solution-fixed sections. Expression and localization of H3.5 were compared with patient background, germinal stage, and PCNA expression. In testes of patients with normal spermatogenesis, differentially expressed H3.5 was specifically localized in either spermatogonia or preleptotene/leptotene-stage primary spermatocytes, especially during germinal stages VI-X. In NOA testes, mRNA expression of H3.5 (H3F3C) was significantly reduced compared with other H3 histone family members, and expression of H3.5 was significantly lower than that in OA. Additionally, the number of H3.5-positive germ cells was higher in hypospermatogenesis or late maturation arrest than in early maturation arrest in NOA testes (p < 0.01). A significant positive correlation was observed between H3.5 and PCNA expression (p < 0.05) but not TUNEL-positive cells, and expression of H3.5 was enhanced after hCG-based salvage hormonal therapy. Different from other testis-specific histones, which are often expressed during the histone-to-protamine transition during meiosis, H3.5 was expressed mainly in immature germ cells. H3.5 may play roles in DNA synthesis, but not apoptosis, and its expression is regulated by gonadotropins, indicating that such epigenetic regulations are important in normal spermatogenesis and spermatogenic disorders.

Niobate nanosheet membranes with enhanced stability for nanofiltration.

Niobate nanosheets are assembled into thin membranes by a vacuum filtration. The nanosheet membranes have a dense and stable structure in water via chemical cross-linking and show higher permeance and salt rejection compared with graphene oxide membranes. A water pathway model based on the void structure is presented to explain the membrane performances.

Effects of human chorionic gonadotropin on testicular interstitial tissues in men with non-obstructive azoospermia.

Non-obstructive azoospermia is a severe condition because spermatogenesis per se is disrupted. Although microdissection testicular sperm extraction is the standard therapy for non-obstructive azoospermia, sperm retrieval is unsuccessful in approximately 50% of patients. For these patients, we conducted human chorionic gonadotropin-based salvage hormonal therapy, and sperm retrieval was possible in 10-20% of patients. The objectives of this study were to assess the changes in interstitial lesions in patients with non-obstructive azoospermia and to evaluate the effects of human chorionic gonadotropin on these tissues. Testicular biopsy specimens were obtained from 10 non-obstructive azoospermia patients who failed to obtain spermatozoa and from 10 obstructive azoospermia patients. All non-obstructive azoospermia patients received salvage hormonal therapy after microdissection testicular sperm extraction. Hematoxylin and eosin (H.E.) staining and immunohistochemical staining for steroidogenic acute regulatory protein antibody, the Leydig cell marker, and TE-7 antibody, the fibroblast marker, as well as picrosirius red staining to detect collagen fibers, were performed. We measured interstitial lesions, as Leydig cell area and the other area, with ImageJ software. Interstitial area, excluding Leydig cells, increased up to 12.5% in non-obstructive azoospermia compared with 1.2% in obstructive azoospermia (p < 0.01), which was mainly because of fibrosis with TE-7-positive fibroblasts. The increase in interstitial lesions was correlated with Johnsen scores. Interstitial area, excluding the Leydig cells, decreased by 29% after salvage hormonal therapy (p < 0.05), indicating improvement of interstitial fibrosis in non-obstructive azoospermia. There were no significant difference in total Leydig cell area and size of each Leydig cells between obstructive azoospermia and non-obstructive azoospermia. After the salvage hormonal therapy, a portion of the Leydig cells became hypertrophied and mean diameter of Leydig cell significantly increased (p < 0.01). This study showed anti-fibrotic effects of human chorionic gonadotropin and hypertrophic change of Leydig cells after human chorionic gonadotropin administration.

Factors Prognostic for Survival in Japanese Patients Treated with Sunitinib as First-line Therapy for Metastatic Clear Cell Renal Cell Cancer.

BACKGROUND: Factors predictive of survival have been identified in Western patients with metastatic clear cell renal cell carcinoma (mCCRCC) treated with sunitinib. Less is known, however, about factors predictive of survival in Japanese patients. This study evaluated factors prognostic of survival in Japanese patients with mCCRCC treated with first-line sunitinib. MATERIALS AND METHODS: This retrospective study evaluated 46 consecutive Japanese mCCRCC patients treated with sunitinib as first line therapy. Clinical and biochemical markers associated with progression-free survival (PFS) were analyzed, with prognostic factors selected by uni- and multivariate Cox regression analyses. RESULTS: Univariate analysis showed that factors significantly associated with poor PFS included Memorial Sloan-Kettering Cancer Center poor risk scores, International Metastatic RCC Database Consortium poor risk and high (>0.5 mg/dl) serum C-reactive protein (CRP) concentrations (p<0.001 each). Multivariate analysis showed that high serum CRP was independently associated with poorer PFS (p=0.040). Six month disease control rate (complete response, partial response and stable disease) in response to sunitinib was significantly higher in patients with normal (≤0.5 mg/dl) than elevated baseline CRP (p<0.001). CONCLUSIONS: CRP is a significant independent predictor of PFS for Japanese patients with mCCRCC treated with first-line sunitinib. Pretreatment CRP concentration may be a useful biomarker predicting response to sunitinib treatment.

Clinical significance of ERG rearrangement subtype and its association with increased p53 expression in Japanese and German prostate cancer.

UNLABELLED: This study investigated differences in prevalence of the androgen-regulated transmembrane protease serine 2 (TMPRSS2) and ETS transcription factor family member, v-ets erythroblastosis virus E26 oncogene homolog (ERG) fusion gene (TMPRSS2-ERG fusions) in clinically localized prostate cancer Japanese and German patients. A total of 105 specimens, including 69 Japanese and 36 German patients, were collected. The status of TMPRSS2-ERG fusion was determined by fluorescence in situ hybridization, and correlations of the TMPRSS2-ERG fusion with clinicopathological characteristics and immunohistochemistry were studied. Gene fusions were identified in 20% (14/69) of Japanese and 53% (19/36) of German patients (P < 0.001). The difference in the type of gene fusion between the two ethnic groups was statistically significant (P=0.024). Overexpression of ERG protein was significantly associated with gene fusion. Biochemical recurrence was significantly higher in patients with ERG overexpression than in those without, and not related to TMPRSS2-ERG fusion status. Interestingly, two types of gene fusions (deletion and increase of copy number) were significantly associated with increased p53 expression (P = 0.005). Association of specific gene fusions harboring higher genomic alterations with p53 expression levels suggests that p53 mutation might drive more aggressive arrangements of TMPRSS2-ERG fusion in prostate cancer. KEYWORDS: ERG, p53, prostate cancer, TMPRSS2-ERG fusion.

MicroRNA regulons in tumor microenvironment.

Cancer initiation and progression are defined by the behavior of cancer cells per se and the development of tumor tissues, both of which are modulated by crosstalk between cancer cells and the surrounding microenvironment. Advances in cancer research have highlighted the significance of constant evolution of the tumor microenvironment, leading to tumor formation, metastasis and refractoriness to therapy. MicroRNAs (miRNAs) are small non-coding RNAs that function as major players of posttranscriptional gene regulation in diverse biological processes. They function as both tumor suppressors and promoters in many aspects of the autonomous behavior of cancer cells. Theoretically, dysfunction in the gene regulatory networks of cancer cells is one of the major driving forces for alterations of ostensibly normal surrounding cells. In this context, the core targets of miRNAs, termed miRNA regulons, are currently being expanded to include various modulators of the tumor microenvironment. Recent advances have highlighted two important roles played by miRNAs in the evolution of tumor microenvironments: miRNAs in tumor cells transform the microenvironment via non-cell-autonomous mechanisms, and miRNAs in neighboring cells stabilize cancer hallmark traits. These observations epitomize the distal and proximal functions of miRNAs in tumor microenvironments, respectively. Such regulation by miRNAs affects tumor angiogenesis, immune invasion and tumor-stromal interactions. This review summarizes recent findings on the mechanisms of miRNA-mediated regulation of tumor microenvironments, with a perspective on the design of therapeutic interventions.

Expression of testicular androgen receptor in non-obstructive azoospermia and its change after hormonal therapy.

Several trials aimed at improving the sperm retrieval from men with non-obstructive azoospermia (NOA) by optimizing intratesticular testosterone (ITT) have reported partial responses, however, an appropriate level of ITT has not been identified. In this study, we examined the expression of the testicular androgen receptor (AR) in NOA and investigated its correlation with clinical and pathological parameters. Expression of the testicular AR was investigated in 52 men with NOA and 22 men with obstructive azoospermia (OA). Twenty-two patients for whom sperm retrieval failed during microdissection testicular sperm extraction (micro-TESE) were enrolled in hormonal therapy using hCG with or without recombinant human follicle stimulating hormone (rhFSH) prior to a second micro-TESE. Sertoli cells were identified by vimentin immunostaining, and positivity in Sertoli cells was used as the AR index. AR immunostaining was robust in the nuclei of Sertoli cells [Sertoli cell androgen receptor (SCAR)] in both OA and NOA. The mean AR index in NOA was significantly higher than that in OA (p < 0.05). In NOA patients, there was no correlation between the AR index and the clinical parameters, whereas the AR index of early maturation arrest (MA) was significantly lower than that of Sertoli cell only, late MA and hypospermatogenesis (p < 0.05). A significant increase in the AR index after salvage hormonal therapy was shown, particularly when using rhFSH. The AR index in patients from whom spermatozoa could be retrieved at the second micro-TESE increased significantly after hormonal therapy. In human testes, the expression of AR is dominant in Sertoli cells, and the expression of SCAR is upregulated by FSH. Germ cell maturation, especially during spermatogonia to spermatocyte stage, has been shown to be SCAR-dependent. Taken together, the results indicate that SCAR elevation is closely associated with sperm retrieval after hormonal therapy and that FSH-based hormonal therapy is potentially effective in NOA men with MA.

Clinicopathological features of mucoepidermoid carcinoma.

OBJECTIVE: We aimed to examine the clinical usefulness of a new World Health Organization classification scheme for salivary gland mucoepidermoid carcinoma, and to identify the factors most strongly associated with prognosis and outcome. METHODS: The clinicopathological features of 45 patients who received treatment for mucoepidermoid carcinoma between 1986 and 2010 were retrospectively investigated. RESULTS: The overall disease-specific 5-year survival rate was 81.8 per cent. The rate for patients with low-grade tumours (92.5 per cent) was significantly higher than that for patients with intermediate or high-grade tumours (52.2 per cent). Univariate analysis revealed that five factors were significantly associated with five-year survival: age, tumour stage classification, lymph node status, histological grade and treatment method. Four factors were significant in multivariate analysis: age, sex, tumour stage classification and lymph node status. CONCLUSION: The new World Health Organization classification was useful in predicting disease progression in patients with mucoepidermoid carcinoma. Patients with high-grade tumours or other prognostic factors positively associated with disease progression should be carefully evaluated and monitored.

Polymeric ion-gels containing an amino acid ionic liquid for facilitated CO2 transport media.

Novel ion-gel films containing an amino acid ionic liquid were fabricated by free radical polymerization of vinyl monomers. These high strength materials demonstrated superior CO2 permeability and separation performance.

Effect of substituting brown rice for corn on lactation and digestion in dairy cows fed diets with a high proportion of grain.

The effects of the substitution of brown rice (Oryza sativa L.; BR) for corn (Zea mays L.) in ensiled total mixed ration (TMR) that had a high proportion of grain on feed intake, lactation performance, ruminal fermentation, digestion, and N utilization were evaluated. Nine multiparous Holstein cows (51 ± 9 d in milk) were used in a replicated 3 × 3 Latin square design with 3 dietary treatments: a diet containing 0, 20, or 40% steam-flaked BR and 40, 20, or 0% steam-flaked corn (dry matter basis). Cows were fed ad libitum an ensiled TMR consisting of 40.7% alfalfa silage, 11.8% grass silage, 7.1% soybean meal, and 40.0% steam-flaked grain (dry matter basis). The ensiled TMR was prepared by baling fresh TMR, and then sealed by a bale wrapper and stored outdoors at 5 to 30 °C for over 6 mo. Dry matter intake and milk yield were lower for cows fed 40% BR than for cows fed 40% corn. The ruminal pH and total volatile fatty acid concentrations were not affected by dietary treatment. The ruminal ammonia-N concentration decreased as the percentage of BR in the diets was elevated. The proportion of acetate decreased, and that of propionate and butyrate increased with the increasing levels of BR. Plasma urea-N concentrations was lower and glucose and insulin concentrations were higher for cows fed 40% BR than for cows fed 40% corn. The whole-tract apparent digestibility of dry matter, organic matter, and starch increased, and the digestibility of neutral detergent fiber and acid detergent fiber decreased with the increasing BR level in the diet, with no dietary effect on crude protein digestion. As a proportion of N intake, the urinary N excretion was lower and the retention of N was higher for cows fed 40% BR than for cows fed 40% corn, with no dietary effect observed on N secretion in milk and fecal N excretion. These results show that substituting BR for corn decreases urinary N losses and improves N utilization, but causes adverse effects on milk production when cows are fed high-grain diets at 40% of dietary dry matter.

The effect of human chorionic gonadotropin-based hormonal therapy on intratesticular testosterone levels and spermatogonial DNA synthesis in men with non-obstructive azoospermia.

The use of hormonal therapy in men with non-obstructive azoospermia (NOA) is controversial because no information is available on how it affects intratesticular testosterone (ITT) levels and spermatogenic cells. The aim of this study was to investigate the ITT level and spermatogonial DNA synthesis, as determined by proliferating cell nuclear antigen (PCNA) expression, before and after human chorionic gonadotropin (hCG)-based hormonal therapy in men with NOA. Twenty patients who failed sperm retrieval procedures using microdissection testicular sperm extraction (micro-TESE) were enrolled in hCG-based hormonal therapy prior to a second micro-TESE. The patients' ITT levels were determined from testicular fluid obtained during the micro-TESE. Spermatogonial PCNA expression was determined by immunohistochemical analysis, and the PCNA labelling index (PCNA-LI) was calculated. During the second micro-TESE, spermatozoa were successfully retrieved from three (15%) of the men who had been treated with hormonal therapy. PCNA-positive cells were predominantly in the spermatogonia and primary spermatocytes, and PCNA-LI was significantly increased after the hormonal therapy. A significant increase in the ITT levels before and after the hormonal therapy (p < 0.0001, 273.6 ± 134.4 and 1348.1 ± 505.4 ng/mL respectively). The sperm-positive group showed a significantly lower basal ITT level compared with the sperm-negative group (p < 0.05). There was a marked increase in the PCNA-LI levels of men treated with both recombinant human follicle-stimulating hormone and hCG. In addition, there was a significant negative association between the increase in PCNA-LI and basal ITT levels at the initial micro-TESE (p < 0.05), but not the stimulated ITT level at the second micro-TESE. HCG-based hormonal therapy significantly raises the ITT level and stimulates spermatogonial DNA synthesis, potentially improving spermatogenesis. ITT optimization plays, at least in part, an important role for stimulating spermatogenesis in men with NOA.

Small cell carcinoma of the head and neck: report of three cases.

OBJECTIVE: Small cell carcinoma of the head and neck is rare and has unique histopathological characteristics that make it difficult to diagnose and treat. In this report, the Japanese Lung Cancer Treatment Guidelines were adapted to treat three patients with small cell carcinoma of the head and neck, and outcomes evaluated. METHODS: There was one case each of stage I small cell carcinoma of the nasal cavity, stage IV-B small cell carcinoma of the ethmoid sinus, and stage IV-A small cell carcinoma of the submandibular gland. All patients underwent chemoradiotherapy and achieved a partial response. RESULTS: Only case one underwent surgery after chemoradiotherapy; 31 months after treatment, this patient had suffered no recurrence. Case two died three months after treatment due to bone marrow metastasis. Case three had experienced no progression after 12 months of follow up. CONCLUSION: In this small patient series, short-term results were equivalent to or better than usual treatment outcomes for small cell carcinoma of the lung.

Roles of M2 and M3 muscarinic receptors in the generation of rhythmic motor activity in mouse small intestine.

BACKGROUND: The roles of M2 and M3 muscarinic receptor subtypes in the regulation of gut motor activity were investigated. METHODS: We simultaneously recorded changes in the intraluminal pressure (IP) and longitudinal tension (LT) in small intestinal segments from M2 or M3 receptor knockout (KO) and wild-type (WT) mice. KEY RESULTS: In the WT preparations, luminal distension induced a continuous rhythmic contractile activity that was characterized by synchronous rises in IP and LT, occurring periodically at a constant interval. Tetrodotoxin completely abolished the response, whereas atropine either abolished or attenuated it. In the majority of the M2 KO preparations, however, no rhythmic activity was observed in response to the luminal distention, even though networks of enteric neurons and interstitial cells of Cajal (ICC) seemed to be intact. Where rhythmic activity did occur in M2 KO preparations, it was atropine resistant. In the M3 KO preparations, the IP and LT were synchronously changed by the luminal distention, but the changes occurred at irregular intervals. The W/W(v) mutant preparations, which lack ICC in the myenteric plexus (ICC-MY), showed results similar to those of the M3 KO preparations. In some of the M2 /M3 double-KO preparations, rhythmic activity was not observed, but in the others, an atropine-resistant rhythmicity appeared. CONCLUSIONS & INFERENCES: These results suggest that M2 and M3 muscarinic receptors differentially regulate the intestinal motor activity: M2 receptors play an essential role in the generation of rhythmic motor activity, and M3 receptors have a modulatory role in controlling the periodicity of the rhythmic activity together with the ICC-MY.

Surgical repair of pelvic-floor prolapse: lessons learned from longitudinal follow-up of quality-of-life survey.

BACKGROUND: Scant evidence has been reported on the evaluation of quality-of-life (QOL) in patients who had undergone surgical treatment due to pelvic floor prolapse including cystocele. The aim of this study is to evaluate the impact of surgical intervention on patients' QOL before and after surgery. METHODS: Between 1997 and 2007, 135 patients (median age: 66.6 years) with pelvic floor prolapse including cystocele underwent bladder neck suspension with anterior/posterior colporrhaphy. The follow-up period was 39.6 months. Seventy-two patients (53 %) had urinary incontinence. The cystocele was graded as mild (grade 2), moderate (grade 3), and severe (grade 4) in 35, 60, and 40, respectively, according to the Baden-Walker classification. A urodynamic study was performed in 69 patients (51 %) who had obstructive symptoms with 100 ml or more of postvoid residual urine. Postoperative QOL was longitudinally assessed in 114 patients by scoring three disease-specific items (sensation of vaginal bulging, obstructive symptoms, urinary incontinence), and one overall health-related QOL (HR-QOL), and compared with corresponding baseline scores. RESULTS: A longitudinal study demonstrated that a significant improvement in these symptoms was sustained at a median follow-up of 62.2 months. HR-QOL was significantly associated with vitality assessed by SF 36 (p = 0.036). Multivariate analysis revealed that update urinary incontinence, pre-operative HR-QOL was independent prognostic factors for predicting postoperative patient's satisfaction. CONCLUSIONS: Although surgical repair of pelvic floor prolapse can achieve acceptable results with intermediate-term durability as well as improving the QOL, preoperative patients' HR-QOL may be considered in the decision making process for treatment.

Methylation level of the RASSF1A promoter is an independent prognostic factor for clear-cell renal cell carcinoma.

BACKGROUND: Ras association domain family 1A (RASSF1A) is a tumor suppressor that regulates the cell cycle, apoptosis, and microtubule stability. The association between the methylation levels of RASSF1A and the prognosis of clear-cell renal cell carcinoma (CCRCC) remains unclear. Therefore, we investigated this relationship to determine the prognostic value of RASSF1A methylation levels for CCRCC. PATIENTS AND METHODS: The study comprised 179 Japanese patients who underwent radical or partial nephrectomy for CCRCC. The methylation level of 5' CpG islands in the RASSF1A was evaluated using combined bisulfite restriction analysis and bisulfite sequencing. RESULTS: High levels of methylation in the RASSF1A promoter were significantly more frequent in grade 3 compared with grade 1 or 2 tumors (P = 0.028) and in patients with stage III or IV compared with patients with stage I or II (P = 0.043). Patients with high methylation levels had a significantly less favorable prognosis compared with those with low methylation levels (P = 0.040). Higher methylation levels were independently associated with a poor prognosis following multivariate analysis (P = 0.0053). CONCLUSION: These results indicate that quantitative promoter methylation levels of the RASSF1A gene may be a useful marker to predict the prognosis of CCRCC.

Evidence that TASK1 channels contribute to the background current in AH/type II neurons of the guinea-pig intestine.

Neurons that have AH (designation of neurons with a prominent and prolonged after hyperpolarizing potential that follows the action potential) electrophysiological characteristics and type II morphology (AH/type II neurons) are the first neurons in reflex circuits in the small intestine. Thus, the state of excitation of these neurons strongly influences the properties of enteric reflexes. The resting outward current in the type II neurons is reduced, causing depolarization and increased excitability, when protein kinase C (PKC) or synaptic inputs are activated, suggesting that regulation of background channels is an important determinant of the state of excitability of these neurons. However, the channels that carry the background current are not yet identified. We used intracellular microelectrodes to record from myenteric AH/type II neurons of the guinea-pig ileum, immunohistochemistry to localize channels and reverse transcriptase-polymerase chain reaction (RT-PCR) to characterize channel transcripts. The blockers of TASK1 channels, bupivacaine (1 mM) and methanandamide (10 muM), depolarized AH/type II neurons by 11.6 mV and 7.9 mV, respectively, and increased resting input resistance by about 30%. The reversal potential determined for the effect of bupivacaine was -92 mV, indicating that bupivacaine acts at K(+) channels, without significant action on other channel types that are open at rest. The membrane potential of type II neurons was depolarized by acidification to pH 6.4, but this depolarization was associated with decreased input resistance and was not reduced by bupivacaine. Thus an unidentified current that is activated by reduced pH masks effects on TASK channels. Slow excitatory post-synaptic potentials in the neurons were reduced in amplitude by methanandamide, suggesting that they are generated in part by closure of TASK1 channels. TASK1 immunoreactivity occurred in all type II neurons (determined by double labeling for IB4 and NeuN), but no type II neurons were immunoreactive for TASK2 or TASK3. These latter channels were localized to non-type II neurons. Transcripts for TASK1, TASK2, TASK3 and other two-pore-domain potassium channels were found in ganglion extracts. It is concluded that TASK1 channels contribute to the resting outward current in AH/type II neurons, and that neurotransmitters that evoke slow depolarizations in these neurons do so through the closure of resting K(+) channels that include TASK1 channels.

Tachykinins and their functions in the gastrointestinal tract.

In the gastrointestinal tract, tachykinins are peptide neurotransmitters in nerve circuits that regulate intestinal motility, secretion, and vascular functions. Tachykinins also contribute to transmission from spinal afferents that innervate the gastrointestinal tract and have roles in the responses of the intestine to inflammation. Tachykinins coexist with acetylcholine, the primary transmitter of excitatory neurons innervating the muscle, and act as a co-neurotransmitter of excitatory neurons. Excitatory transmission is mediated through NK1 receptors (primarily on interstitial cells of Cajal) and NK2 receptors on the muscle. Tachykinins participate in slow excitatory transmission at neuro-neuronal synapses, through NK1 and NK3 receptors, in both ascending and descending pathways affecting motility. Activation of receptors (NK1 and NK2) on the epithelium causes fluid secretion. Tachykinin receptors on immune cells are activated during inflammation of the gut. Finally, tachykinins are released from the central terminals of gastrointestinal afferent neurons in the spinal cord, particularly in nociceptive pathways.

Roles of M2 and M3 muscarinic receptors in cholinergic nerve-induced contractions in mouse ileum studied with receptor knockout mice.

BACKGROUND AND PURPOSE: The functional roles of M(2) and M(3) muscarinic receptors in neurogenic cholinergic contractions in gastrointestinal tracts remain to be elucidated. To address this issue, we studied cholinergic nerve-induced contractions in the ileum using mutant mice lacking M(2) or M(3) receptor subtypes. EXPERIMENTAL APPROACH: Contractile responses to transmural electrical (TE) stimulation were isometrically recorded in ileal segments from M(2)-knockout (KO), M(3)-KO, M(2)/M(3)-double KO, and wild-type mice. KEY RESULTS: TE stimulation at 2-50 Hz frequency-dependently evoked a fast, brief contraction followed by a slower, longer one in wild-type, M(2)-KO or M(3)-KO mouse preparations. Tetrodotoxin blocked both the initial and later contractions, while atropine only inhibited the initial contractions. The initial cholinergic contractions were significantly greater in wild-type than M(2)-KO or M(3)-KO mice; the respective mean amplitudes at 50 Hz were 91, 74 and 68 % of 70mM K(+)-induced contraction. Pretreatment with pertussis toxin blocked the cholinergic contractions in M(3)-KO but not in M(2)-KO mice. Cholinergic contractions also remained in wild-type preparations, but their sizes were reduced by 20-30 % at 10-50 Hz. In M(2)/M(3)-double KO mice, TE stimulation evoked only slow, noncholinergic contractions, which were significantly greater in sizes than in any of the other three mouse strains. CONCLUSION AND IMPLICATIONS: These results demonstrate that M(2) and M(3) receptors participate in mediating cholinergic contractions in mouse ileum with the latter receptors assuming a greater role. Our data also suggest that the lack of both M(2) and M(3) receptors causes upregulation of noncholinergic excitatory innervation of the gut smooth muscle.