Ambroxol-Enhanced Frequency and Amplitude of Beating Cilia Controlled by a Voltage-Gated Ca2+ Channel, Cav1.2, via pHi Increase and [Cl-]i Decrease in the Lung Airway Epithelial Cells of Mice.

Ambroxol (ABX), a frequently prescribed secretolytic agent which enhances the ciliary beat frequency (CBF) and ciliary bend angle (CBA, an index of amplitude) by 30%, activates a voltage-dependent Ca2+ channel (CaV1.2) and a small transient Ca2+ release in the ciliated lung airway epithelial cells (c-LAECs) of mice. The activation of CaV1.2 alone enhanced the CBF and CBA by 20%, mediated by a pHi increasei and a [Cl-]i decrease in the c-LAECs. The increase in pHi, which was induced by the activation of the Na+-HCO3- cotransporter (NBC), enhanced the CBF (by 30%) and CBA (by 15-20%), and a decrease in [Cl-]i, which was induced by the Cl- release via anoctamine 1 (ANO1), enhanced the CBA (by 10-15%). While a Ca2+-free solution or nifedipine (an inhibitor of CaV1.2) inhibited 70% of the CBF and CBA enhancement using ABX, CaV1.2 enhanced most of the CBF and CBA increases using ABX. The activation of the CaV1.2 existing in the cilia stimulates the NBC to increase pHi and ANO1 to decrease the [Cl-]i in the c-LAECs. In conclusion, the pHi increase and the [Cl-]i decrease enhanced the CBF and CBA in the ABX-stimulated c-LAECs.

Adjunctive use of oral MAF is associated with no disease progression or mortality in hospitalized patients with COVID-19 pneumonia: The single-arm COral-MAF1 prospective trial.

Based on a growing body of evidence that a dysregulated innate immune response mediated by monocytes/macrophages plays a key role in the pathogenesis of COVID-19, a clinical trial was conducted to investigate the therapeutic potential and safety of oral macrophage activating factor (MAF) plus standard of care (SoC) in the treatment of hospitalized patients with COVID-19 pneumonia. Ninety-seven hospitalized patients with confirmed COVID-19 pneumonia were treated with oral MAF and a vitamin D3 supplement, in combination with SoC, in a single-arm, open label, multicentre, phase II clinical trial. The primary outcome measure was a reduction in an intensive care unit transfer rate below 13% after MAF administration. At the end of the study, an additional propensity score matching (PSM) analysis was performed to compare the MAF group with a control group treated with SoC alone. Out of 97 patients treated with MAF, none needed care in the ICU and/or intubation with mechanical ventilation or died during hospitalization. Oxygen therapy was discontinued after a median of nine days of MAF treatment. The median length of viral shedding and hospital stay was 14 days and 18 days, respectively. After PSM, statistically significant differences were found in all of the in-hospital outcomes between the two groups. No mild to serious adverse events were recorded during the study. Notwithstanding the limitations of a single-arm study, which prevented definitive conclusions, a 21-day course of MAF treatment plus SoC was found to be safe and promising in the treatment of hospitalized adult patients with COVID-19 pneumonia. Further research will be needed to confirm these preliminary findings.

[The Design Principle of Allostatic Mechanisms: The Free Energy Principle].

Homeostasis is maintained by neural reflex controls, which attempt to maintain the body's internal organs within a predetermined range. However, when deviations are significant, homeostasis may not be restored by these standard controls. Therefore, a function called allostasis is necessary to maximize the energy efficiency of bodily functions, anticipate changing needs (e.g., a future increase in body temperature or oxygen demand), and regulate the body's state before dyshomeostasis occurs. In terms of the free energy principle (FEP), the expectation to survive (i.e., to stay within established confines and to maintain homeostatic integrity over time) is realized by a hierarchical prediction mechanism. Allostasis requires the integration of exteroceptive, interoceptive, and proprioceptive signals. In this paper, the neural mechanism of allostasis is discussed in terms of the FEP.

Ambroxol-enhanced ciliary beating via voltage-gated Ca2+ channels in mouse airway ciliated cells.

Ambroxol (ABX) facilitates the mucociliary clearance (MC) by enhancing ciliary beating in airways. In this study, we focused on airway ciliary beating enhanced by ABX. However, little is known about the ABX-stimulated Ca2+ signalling activating airway ciliary beating. Airway ciliated cells isolated from mice lungs were observed by a high-speed video microscope, and the activities of beating cilia were assessed by CBF (ciliary beat frequency) and CBD (ciliary bend distance, an index of amplitude). ABX (10 µM) enhanced the CBF and CBD by 30%, and the enhancement was inhibited by nifedipine (20 µM, a L-type voltage-gated Ca2+ channel (CaV) inhibitor), or a Ca2+-free solution (approximately 50%). Pre-treatment with BAPTA-AM (10 µM, a chelator of intracellular Ca2+) abolished ABX-stimulated increases in CBF, CBD and [Ca2+]i. Thus, ABX increases [Ca2+]i (intracellular Ca2+ concentration) by stimulating Ca2+ release from the internal stores and nifedipine-sensitive Ca2+ entry. A previous study demonstrated the expression of CaV1.2 in airway cilia. ABX enhanced CBF, CBD and [Ca2+]i even in a high extracellular K+ concentration (155.5 mM), suggesting that it activates CaV1.2 except by depolarization. These enhancements were inhibited by nifedipine. In conclusion, ABX, which increases [Ca2+]i by stimulating Ca2+ release from internal stores and Ca2+ entry through CaV1.2s, enhanced CBF and CBD in airway ciliated cells. ABX is a novel agonist that modulates CaV1.2 of airway beating cilia to enhance CBF and CBD.

Enhancement of airway ciliary beating mediated via voltage-gated Ca2+ channels/α7-nicotinic receptors in mice.

Acetylcholine (ACh), which activates muscarinic ACh receptors (mAChRs) and nicotinic ACh receptors (nAChRs), enhances airway ciliary beating by increasing the intracellular Ca2+ concentration ([Ca2+]i). The mechanisms enhancing airway ciliary beating by nAChRs have remained largely unknown, although those by mAChRs are well understood. In this study, we focused on the effects of α7-nAChRs and voltage-gated Ca2+ channels (CaVs) on the airway ciliary beating. The activities of ciliary beating were assessed by frequency (CBF, ciliary beat frequency) and amplitude (CBD, ciliary bend distance) measured by high-speed video microscopy. ACh enhanced CBF and CBD by 25% mediated by an [Ca2+]i increase stimulated by mAChRs and α7-nAChRs (a subunit of nAChR) in airway ciliary cells of mice. Experiments using PNU282987 (an agonist of α7-nAChR) and MLA (an inhibitor of α7-nAChR) revealed that CBF and CBD enhanced by α7-nAChR are approximately 50% of those enhanced by ACh. CBF, CBD, and [Ca2+]i enhanced by α7-nAChRs were inhibited by nifedipine, suggesting activation of CaVs by α7-nAChRs. Experiments using a high K+ solution with/without nifedipine (155.5 mM K+) showed that the activation of CaVs enhances CBF and CBD via an [Ca2+]i increase. Immunofluorescence and immunoblotting studies demonstrated that Cav1.2 and α7-nAChR are expressed in airway cilia. Moreover, IL-13 stimulated MLA-sensitive increases in CBF and CBD in airway ciliary cells, suggesting an autocrine regulation of ciliary beating by CaV1.2/α7-nAChR/ACh. In conclusion, a novel Ca2+ signalling pathway in airway cilia, CaV1.2/α7-nAChR, enhances CBF and CBD and activates mucociliary clearance maintaining healthy airways.

Degalactosylated Whey Protein Suppresses Inflammatory Responses Induced by Lipopolysaccharide in Mice.

The effects of degalactosylated whey protein on lipopolysaccharide (LPS)-induced inflammatory responses in mice were observed in comparison with intact whey protein. Intraperitoneal administration of both intact and degalactosylated whey proteins for 5 days did not affect body weight and food intake in mice. On day 6, intraperitoneal administration of LPS induced a marked decrease in body weight 4 h later. The LPS-induced decrease in body weight was significantly suppressed by the administration of degalactosylated whey protein, but not intact whey protein. Administration of LPS also significantly increase plasma tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) levels, which were significantly suppressed by the administration of degalactosylated whey protein, but not intact whey protein. Moreover, the application of degalactosylated whey protein to RAW264.7 cells significantly reduced mRNA expression of toll-like receptor 4 (TLR4) and significantly increased mRNA expression of mitogen-activated protein kinase phosphatase-1 (MKP-1). The marked increased expression of TNF-α and IL-1ß in response to LPS in RAW264.7 cells was significantly suppressed by the application of degalactosylated whey protein. These results suggest that degalactosylated whey protein suppresses the effects of LPS in part by decreasing in TLR4 and increasing in MKP-1.

Minimum biological domain of xenin-25 required to induce anion secretion in the rat ileum.

Xenin-25 has a variety of physiological functions in the gastrointestinal tract, including ion transport and motility. Xenin-25 and neurotensin show sequence homology, especially near their C-terminal regions. The sequence similarity between xenin-25 and neurotensin indicates that the effects of xenin-25 is mediated by the neurotensin receptor but some biological actions of xenin-25 are independent. We have previously reported that xenin-25 modulates intestinal ion transport and colonic smooth muscle activity. However, minimal biological domain of xenin-25 to induce ion transport was not clear. To improve the mechanistic understanding of xenin-25 and to gain additional insights into the functions of xenin-25, the present study was designed to determine the minimal biological domain of xenin-25 required for ion transport in the rat ileum using various truncated xenin fragments and analogues in an Ussing chamber system. The present results demonstrate that the minimum biological domain of xenin-25 to induce Cl-/HCO3- secretion in the ileum contains the C-terminal pentapeptide. Furthermore, Arg at position 21 is important to retain the biological activity of xenin-25 and induces Cl-/HCO3- secretion in the rat ileum.

Nitric oxide synthesis stimulated by arachidonic acid accumulation via PPARα in acetylcholine-stimulated gastric mucous cells.

NEW FINDINGS: What is the central question of this study? Arachidonic acid (AA) stimulates NO production in antral mucous cells without any increase in [Ca2+ ]i . Given that the intracellular AA concentration is too low to measure, the relationship between AA accumulation and NO production remains uncertain. Is AA accumulation a key step for NO production? What is the main finding and its importance? We demonstrated that AA accumulation is a key step for NO production. The amount of AA released could be measured using fluorescence-HPLC. The intracellular AA concentration was maintained at < 1 µM. Nitric oxide is produced by AA accumulation in antral mucous cells, not as a direct effect of [Ca2+ ]i . ABSTRACT: In the present study, we demonstrate that NO production is stimulated by an accumulation of arachidonic acid (AA) mediated via peroxisome proliferation-activated receptor α (PPARα) and that the NO produced enhances Ca2+ -regulated exocytosis in ACh-stimulated antral mucous cells. The amount of AA released from the antral mucosa, measured by fluorescence high-performance liquid chromatography (F-HPLC), was increased by addition of ionomycin (10 µM) or ACh, suggesting that AA accumulation is stimulated by an increase in [Ca2+ ]i . The AA production was inhibited by an inhibitor of cytosolic phospholipase A2 (cPLA2-inhα). GW6471 (a PPARα inhibitor) and cPLA2-inhα inhibited NO synthesis stimulated by ACh. Moreover, indomethacin, an inhibitor of cyclooxygenase, stimulated AA accumulation and NO production. However, acetylsalicylic acid did not stimulate AA production and NO synthesis. An analogue of AA (AACOCF3) alone stimulated NO synthesis, which was inhibited by GW6471. In antral mucous cells, indomethacin enhanced Ca2+ -regulated exocytosis by increasing NO via PPARα, and the enhancement was abolished by GW6471 and cPLA2-inhα. Thus, AA produced via PLA2 activation is the key step for NO synthesis in ACh-stimulated antral mucous cells and plays important roles in maintaining antral mucous secretion, especially in Ca2+ -regulated exocytosis.

The effect of Xenin25 on spontaneous circular muscle contractions of rat distal colon in vitro.

Xenin25 has a variety of physiological functions in the Gastrointestinal (GI) tract, including ion transport and motility. However, the motility responses in the colon induced by Xenin25 remain poorly understood. Therefore, the effect of Xenin25 on the spontaneous circular muscle contractions of the rat distal colon was investigated using organ bath chambers and immunohistochemistry. Xenin25 induced the inhibition followed by postinhibitory spontaneous contractions with a higher frequency in the rat distal colon. This inhibitory effect of Xenin25 was significantly suppressed by TTX but not by atropine. The inhibitory time (the duration of inhibition) caused by Xenin25 was shortened by the NTSR1 antagonist SR48692, the NK1R antagonist CP96345, the VPAC2 receptor antagonist PG99-465, the nitric oxide-sensitive guanylate-cyclase inhibitor ODQ, and the Ca2+ -dependent K+ channel blocker apamin. The higher frequency of postinhibitory spontaneous contractions induced by Xenin25 was also attenuated by ODQ and apamin. SP-, NOS-, and VIP-immunoreactive neurons were detected in the myenteric plexus (MP) of the rat distal colon. Small subsets of the SP-positive neurons were also Calbindin positive. Most of the VIP-positive neurons were also NOS positive, and small subsets of the NK1R-positive neurons were also VIP positive. Based on the present results, we propose the following mechanism. Xenin25 activates neuronal NTSR1 on the SP neurons of IPANs, and transmitters from the VIP and apamin-sensitive NO neurons synergistically inhibit the spontaneous circular muscle contractions via NK1R. Subsequently, the postinhibitory spontaneous contractions are induced by the offset of apamin-sensitive NO neuron activation via the interstitial cells of Cajal. In addition, Xenin25 also activates the muscular NTSR1 to induce relaxation. Thus, Xenin25 is considered to be an important modulator of post prandial circular muscle contraction of distal colon since the release of Xenin25 from enteroendocrine cells is stimulated by food intake.

Microbiota-gut-brain axis: enteroendocrine cells and the enteric nervous system form an interface between the microbiota and the central nervous system.

The microbiota-gut-brain axis transmits bidirectional communication between the gut and the central nervous system and links the emotional and cognitive centers of the brain with peripheral gut functions. This communication occurs along the axis via local, paracrine, and endocrine mechanisms involving a variety of gut-derived peptide/amine produced by enteroendocrine cells. Neural networks, such as the enteric nervous system, and the central nervous system, including the autonomic nervous system, also transmit information through the microbiota-gut-brain axis. Recent advances in research have described the importance of the gut microbiota in influencing normal physiology and contributing to disease. We are only beginning to understand this bidirectional communication system. In this review, we summarize the available data supporting the existence of these interactions, highlighting data related to the contribution of enteroendocrine cells and the enteric nervous system as an interface between the gut microbiota and brain.

Intracellular Cl- Regulation of Ciliary Beating in Ciliated Human Nasal Epithelial Cells: Frequency and Distance of Ciliary Beating Observed by High-Speed Video Microscopy.

Small inhaled particles, which are entrapped by the mucous layer that is maintained by mucous secretion via mucin exocytosis and fluid secretion, are removed from the nasal cavity by beating cilia. The functional activities of beating cilia are assessed by their frequency and the amplitude. Nasal ciliary beating is controlled by intracellular ions (Ca2+, H+ and Cl-), and is enhanced by a decreased concentration of intracellular Cl- ([Cl-]i) in ciliated human nasal epithelial cells (cHNECs) in primary culture, which increases the ciliary beat amplitude. A novel method to measure both ciliary beat frequency (CBF) and ciliary beat distance (CBD, an index of ciliary beat amplitude) in cHNECs has been developed using high-speed video microscopy, which revealed that a decrease in [Cl-]i increased CBD, but not CBF, and an increase in [Cl-]i decreased both CBD and CBF. Thus, [Cl-]i inhibits ciliary beating in cHNECs, suggesting that axonemal structures controlling CBD and CBF may have Cl- sensors and be regulated by [Cl-]i. These observations indicate that the activation of Cl- secretion stimulates ciliary beating (increased CBD) mediated via a decrease in [Cl-]i in cHNECs. Thus, [Cl-]i is critical for controlling ciliary beating in cHNECs. This review introduces the concept of Cl- regulation of ciliary beating in cHNECs.

Airway Ciliary Beating Affected by the Pcp4 Dose-Dependent [Ca2+]i Increase in Down Syndrome Mice, Ts1Rhr.

In Ts1Rhr, a Down syndrome model mouse, the airway ciliary beatings are impaired; that is, decreases in ciliary beat frequency (CBF) and ciliary bend angle (CBA, an index of ciliary beat amplitude)). A resumption to two copies of the Pcp4 gene on the Ts1Rhr trisomic segment (Ts1Rhr:Pcp4+/+/-) rescues the decreases in CBF and CBA that occur in Ts1Rhr. In airway cilia, upon stimulation with procaterol (a ß2-agonist), the CBF increase is slower over the time course than the CBA increase because of cAMP degradation by Ca2+/calmodulin-dependent phosphodiesterase 1 (PDE1) existing in the metabolon regulating CBF. In Ts1Rhr, procaterol-stimulated CBF increase was much slower over the time course than in the wild-type mouse (Wt) or Ts1Rhr:Pcp4+/+/-. However, in the presence of 8MmIBMX (8-methoxymethyl isobutylmethyl xanthine, an inhibitor of PDE1) or calmidazolium (an inhibitor of calmodulin), in both Wt and Ts1Rhr, procaterol stimulates CBF and CBA increases over a similar time course. Measurements of cAMP revealed that the cAMP contents were lower in Ts1Rhr than in Wt or in Ts1Rhr:Pcp4+/+/-, suggesting the activation of PDE1A that is present in Ts1Rhr airway cilia. Measurements of the intracellular Ca2+ concentration ([Ca2+]i) in airway ciliary cells revealed that temperature (increasing from 25 to 37 °C) or 4αPDD (a selective transient receptor potential vanilloid 4 (TRPV4) agonist) stimulates a larger [Ca2+]i increase in Ts1Rhr than in Wt or Ts1Rhr:Pcp4+/+/-. In airway ciliary cells of Ts1Rhr, Pcp4-dose dependent activation of TRPV4 appears to induce an increase in the basal [Ca2+]i. In early embryonic day mice, a basal [Ca2+]i increased by PCP4 expressed may affect axonemal regulatory complexes regulated by the Ca2+-signal in Ts1Rhr, leading to a decrease in the basal CBF and CBA of airway cilia.

Enhancement of ciliary beat amplitude by carbocisteine in ciliated human nasal epithelial cells.

OBJECTIVE: Carbocisteine (CCis), a mucoactive agent, is used to improve the symptoms of sinonasal diseases. However, the effect of CCis on nasal ciliary beating remains uncertain. We examined the effects of CCis on ciliary beat distance (CBD, an index of amplitude), and ciliary beat frequency (CBF) in ciliated human nasal epithelial cells (cHNECs) in primary culture. METHODS: The cHNECs were prepared from the nasal tissue resected from patients required surgery for chronic sinusitis (CS) or allergic rhinitis (AR). CBD and CBF were measured using videomicroscopy equipped with a high-speed camera. RESULTS: CCis increased CBD by 30%, but not CBF, and decreased intracellular Cl- concentration ([Cl- ]i ) in cHNECs. The CCis' actions were mimicked by the Cl- -free NO3- solution. In contrast, prior treatment of NPPB (20 µM) or CFTR(inh)-172 (1 µM), which increased [Cl- ]i by 20%, decreased CBF by 10% and CBD by 25% and inhibited the CCis' actions. However, prior treatment of T16Ainh-A01 (10 µM) did not inhibit the CCis' actions, although it decreased [Cl- ]i by 10% and CBD by 15%. Thus, CCis stimulates Cl- channels including cystic fibrosis transmembrane conductance regulator (CFTR). Moreover, CCis enhanced the transport of microbeads driven by the beating cilia in cHNECs. The CCis actions were similar in cHNECs from both types of pateints. CONCLUSION: CCis increased CBD by 30% in cHNECs via an [Cl- ]i decrease stimulated by activation of Cl- channels, including CFTR. CCis may stimulate nasal mucociliary clearance by increasing CBD in patients contracting CS or AR. LEVEL OF EVIDENCE: NA. Laryngoscope, 130:E289-E297, 2020.

Abnormal Sensorimotor Integration in Adults Who Stutter: A Behavioral Study by Adaptation of Delayed Auditory Feedback.

Stuttering is a fluency disorder, partially alleviated during altered auditory feedback, suggesting abnormal sensorimotor integration in adults who stutter (AWS). As weighting of multiple integrating-information sources would be decided based on their reliabilities, the use of external (auditory feedback) and internal information (prediction of sensory consequences) could correlate with speech processing. We hypothesized that abnormal auditory-feedback processing in AWS could be related to decrease in internal processing precision. We used a perceptual-adaptation experiment of delayed auditory feedback (DAF) to verify the hypothesis. Seventeen AWS and 17 adults who do not stutter (ANS) were required to say "ah" and judge the simultaneity between their motor sensations and vocal sounds in each stimulus onset asynchrony (SOA) (0, 25, 50, 75, 100, 125, or 150 ms) after inducing adaptation of DAF (three conditions with 0-, 66-, or 133-ms delay). While no adaptation occurred during the 0 ms condition, perceptual change in simultaneity judgment (adaptation effect) occurred during the 66 and 133 ms conditions. The simultaneity judgments following exposure in each SOA were fitted to the psychometric function in each condition for the AWS and ANS groups. We calculated the µ (signifying the point of subjective simultaneity and adaptation-effect degree) and σ (signifying the detecting precision) of each function and analyzed them by parametric analyses. For the µ, participant groups and adaptation conditions showed a significant interaction; the adaptation effect was greater in the AWS than in the ANS group. Additionally, the µ and σ were only positively correlated in the AWS group. The point of subjective simultaneity for auditory delay by inducing DAF was higher in AWS than in ANS, indicating that perception of simultaneity in AWS was influenced by DAF to a greater extent. Moreover, the significant positive correlation between the µ and σ in AWS showed that the more imprecise the internal auditory processing, the more AWS relied on auditory feedback. It is suggested that the reliability of internal information differed within the AWS group, and AWS with reduced internal reliability appeared to compensate by relying to a great extent on auditory feedback information.

Ciliary beating amplitude controlled by intracellular Cl- and a high rate of CO2 production in ciliated human nasal epithelial cells.

The ciliary transport is controlled by two parameters of the ciliary beating, frequency (CBF) and amplitude. In this study, we developed a novel method to measure both CBF and ciliary bend distance (CBD, an index of ciliary beating amplitude) in ciliated human nasal epithelial cells (cHNECs) in primary culture, which are prepared from patients contracting allergic rhinitis and chronic sinusitis. An application of Cl--free NO3- solution or bumetanide (an inhibitor of Na+/K+/2Cl- cotransport), which decreases intracellular Cl- concentration ([Cl-]i), increased CBD, not CBF, at 37 °C; however, it increased both CBD and CBF at 25 °C. Conversely, addition of Cl- channel blockers (5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and 4-[[4-Oxo-2-thioxo-3-[3-trifluoromethyl]phenyl]-5-thiazolidinylidene]methyl] benzoic acid (CFTR(inh)-172)), which increase [Cl-]i, decreased both CBD and CBF, suggesting that CFTR plays a crucial role for maintaining [Cl-]i in these cells. We speculate that Cl- modulates activities of the molecular motors regulating both CBD and CBF in cHNECs. Moreover, application of the CO2/HCO3--free solution did not change intracellular pH (pHi), and addition of an inhibitor of carbonic anhydrase (acetazolamide) sustained pHi increase induced by the NH4+ pulse, which transiently increased pHi in the absence of acetazolamide. These results indicate that the cHNEC produces a large amount of CO2, which maintains a constant pHi even under the CO2/HCO3--free condition.

Xenin-25 induces anion secretion by activating noncholinergic secretomotor neurons in the rat ileum.

Xenin-25 is a neurotensin-like peptide that is secreted by enteroendocrine cells in the small intestine. Xenin-8 is reported to augment duodenal anion secretion by activating afferent neural pathways. The intrinsic neuronal circuits mediating the xenin-25-induced anion secretion were characterized using the Ussing-chambered, mucosa-submucosa preparation from the rat ileum. Serosal application of xenin-25 increased the short-circuit current in a concentration-dependent manner. The responses were abolished by the combination of Cl--free and HCO3- -free solutions. The responses were almost completely blocked by TTX (10-6 M) but not by atropine (10-5 M) or hexamethonium (10-4 M). The selective antagonists for neurotensin receptor 1 (NTSR1), neurokinin 1 (NK1), vasoactive intestinal polypeptide (VIP) receptors 1 and 2 (VPAC1 and VPAC2, respectively), and capsaicin, but not 5-hydroxyltryptamine receptors 3 and 4 (5-HT3 and 5-HT4), NTSR2, and A803467, inhibited the responses to xenin-25. The expression of VIP receptors (Vipr) in rat ileum was examined using RT-PCR. The Vipr1 PCR products were detected in the submucosal plexus and mucosa. Immunohistochemical staining showed the colocalization of NTSR1 and NK1 with substance P (SP)- and calbindin-immunoreactive neurons in the submucosal plexus, respectively. In addition, NK1 was colocalized with noncholinergic VIP secretomotor neurons. Based on the results from the present study, xenin-25-induced Cl-/ HCO3- secretion is involved in NTSR1 activation on intrinsic and extrinsic afferent neurons, followed by the release of SP and subsequent activation of NK1 expressed on noncholinergic VIP secretomotor neurons. Finally, the secreted VIP may activate VPAC1 on epithelial cells to induce Cl-/ HCO3- secretion in the rat ileum. Activation of noncholinergic VIP secretomotor neurons by intrinsic primary afferent neurons and extrinsic afferent neurons by postprandially released xenin-25 may account for most of the neurogenic secretory response induced by xenin-25. NEW & NOTEWORTHY This study is the first to investigate the intrinsic neuronal circuit responsible for xenin-25-induced anion secretion in the rat small intestine. We have found that nutrient-stimulated xenin-25 release may activate noncholinergic vasoactive intestinal polypeptide (VIP) secretomotor neurons to promote Cl-/ HCO3- secretion through the activation of VIP receptor 1 on epithelial cells. Moreover, the xenin-25-induced secretory responses are mainly linked with intrinsic primary afferent neurons, which are involved in the activation of neurotensin receptor 1 and neurokinin 1 receptor.

Ease of hand rotation during active exploration of views of a 3-D object modulates view generalization.

Active exploration of views of 3-D objects by manually controlling a device, such as a trackball, facilitates subsequent object recognition, suggesting that motor simulation contributes to object recognition. Further, biomechanical constraints, such as range of hand rotation, can affect mental rotation. Thus, the ease with which an object can be rotated by hand may modulate the facilitative effect active exploration through manual control has on object recognition. In our experiment, participants performed two sessions of a view-matching task, with a learning task administered between the two. In the learning task, one group of participants (active group) viewed and explored a novel 3-D object using their hand to rotate a handle attached to a cathode-ray tube monitor. The other group (passive group) observed on the monitor a replay of the movements of the 3-D object as manipulated by an active-group participant. Active-group participants were interviewed to determine the direction they found easiest to rotate their hand. The view-generalization performances were compared between the pre and post sessions. Although we observed a facilitative effect on the view-matching process in both groups, the active group exhibited view-dependent facilitation. The view-generalization range of the active group in the post-session was asymmetric in terms of the rotation direction. Most intriguingly, for most participants, this asymmetric change corresponded to the direction that afforded the easiest hand rotation (ulnar deviation). These findings suggest that the object-recognition process can be affected by ease of hand rotation, which is based on the biomechanical constraints of the wrist joint.

Carbocisteine stimulated an increase in ciliary bend angle via a decrease in [Cl-]i in mouse airway cilia.

Carbocisteine (CCis), a mucoactive agent, is widely used to improve respiratory diseases. This study demonstrated that CCis increases ciliary bend angle (CBA) by 30% and ciliary beat frequency (CBF) by 10% in mouse airway ciliary cells. These increases were induced by an elevation in intracellular pH (pHi; the pHi pathway) and a decrease in the intracellular Cl- concentration ([Cl-]i; the Cl- pathway) stimulated by CCis. The Cl- pathway, which is independent of CO2/HCO3-, increased CBA by 20%. This pathway activated Cl- release via activation of Cl- channels, leading to a decrease in [Cl-]i, and was inhibited by Cl- channel blockers (5-nitro-2-(3-phenylpropylamino) benzoic acid and CFTR(inh)-172). Under the CO2/HCO3--free condition, the CBA increase stimulated by CCis was mimicked by the Cl--free NO3- solution. The pHi pathway, which depends on CO2/HCO3-, increased CBF and CBA by 10%. This pathway activated HCO3- entry via Na+/HCO3- cotransport (NBC), leading to a pHi elevation, and was inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid. The effects of CCis were not affected by a protein kinase A inhibitor (1 µM PKI-A) or Ca2+-free solution. Thus, CCis decreased [Cl-]i via activation of Cl- channels including CFTR, increasing CBA by 20%, and elevated pHi via NBC activation, increasing CBF and CBA by 10%.

Bioconjugation strategy for cell surface labelling with gold nanostructures designed for highly localized pH measurement.

Regulation of intracellular pH is critically important for many cellular functions. The quantification of proton extrusion in different types of cells and physiological conditions is pivotal to fully elucidate the mechanisms of pH homeostasis. Here we show the use of gold nanoparticles (AuNP) to create a high spatial resolution sensor for measuring extracellular pH in proximity of the cell membrane. We test the sensor on HepG2 liver cancer cells and MKN28 gastric cancer cells before and after inhibition of Na+/H+ exchanger. The gold surface conjugation strategy is conceived with a twofold purpose: i) to anchor the AuNP to the membrane proteins and ii) to quantify the local pH from AuNP using surface enhanced Raman spectroscopy (SERS). The nanometer size of the cell membrane anchored sensor and the use of SERS enable us to visualize highly localized variation of pH induced by H+ extrusion, which is particularly upregulated in cancer cells.

Improvement of Diabetes Mellitus Symptoms by Intake of Ninjin'yoeito.

Diabetes mellitus is a well-known common disease and one of the most serious social problems in the worldwide. Although various types of drugs are developed, the number of patients suffering from diabetes mellitus is still increasing. Ninjin'yoeito (NYT) is one of formulas used in Japanese traditional herbal medicines for improving various types of metabolic disorders. However, the effect of NYT on diabetes mellitus has not yet been investigated. In the present study, we tried to clarify the action of NYT on the serum glucose level in streptozotocin (STZ)-induced diabetic mice. We found that intake of NYT decreased the serum glucose level and increased insulin sensitivity in STZ-induced diabetic mice. NYT treatment also improved acidification of the interstitial fluid around skeletal muscles found in STZ-induced diabetic mice, while the interstitial fluid acidification has been reported to cause insulin resistance. Furthermore, in the proximal colon of STZ-induced diabetic mice, NYT treatment showed a tendency to increase the expression of sodium-coupled monocarboxylate transporter 1 (SMCT1), which has ability to absorb weak organic acids (pH buffer molecules) resulting in improvement of the interstitial fluid acidification. Based on these observations, the present study suggests that NYT is a useful formula to improve hyperglycemia and insulin resistance via elevation of interstitial fluid pH in diabetes mellitus, which might be caused by increased absorption of pH buffer molecules (SMCT1 substrates, weak organic acids) mediated through possibly elevated SMCT1 expression in the proximal colon.