Regenerative medicine is a new field based on the use of stem cells to generate biological substitutes and improve tissue functions, restoring damaged tissue with high proliferability and differentiability. It is of interest as a potential alternative to complicated tissue/organ transplantation. Recently, amnion-derived cells have been reported to have multipotent differentiation ability, and these cells have attracted attention as a cell source for cell-transplantation therapy. The amnion possesses considerable advantageous characteristics: the isolated cells can differentiate into all three germ layers; they have low immunogenicity and anti-inflammatory functions; and they do not require the sacrifice of human embryos for their isolation, thus avoiding the current controversies associated with the use of human embryonic stem cells. Moreover, we developed human amniotic cell-sheets using a novel culture surface coated with a noncytotoxic, temperature-responsive elastic protein-based polymer. We also generated a “hyper-dry-amnion”, which has already been applied clinically in the ophthalmological field. Compared to cryopreserved fresh amnion, “hyper-dry-amnion” is easy to handle and has started to bring good results to patients. These materials from the amnion are also expected to open a new field in tissue engineering. Thus, amnion, which had been discarded after parturition, has started to be appreciated as an attractive material in the field of regenerative medicine. In this review, the most recent and relevant clinical and experimental data about the use of amniotic membrane and cells derived from it are described.

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Effects of azimilide, a class III antiarrhythmic drug, on the acetylcholine (ACh) receptor-operated K⁺ current (I_K.ACh) and the delayed rectifier K⁺ current (I_K) were examined in guinea-pig atrial cells using patch-clamp techniques. Effects of azimilide on experimental atrial fibrillation (AF) were also examined in isolated guinea-pig hearts. In single atrial myocytes, azimilide inhibited both the rapid (I_Kr) and slow component of I_K (I_Ks). Azimilide inhibited the I_K.ACh induced by carbachol (CCh, 1 μM), adenosine (10 μM), and intracellular loading of GTPγS (100 μM) in a concentration-dependent manner. The IC₅₀ values of azimilide for inhibiting the CCh-, adenosine-, and GTPγS-induced I_K.ACh were 1.25, 29.1, and 20.9 μM, respectively, suggesting that azimilide inhibits I_K.ACh mainly by blocking the muscarinic receptors. Azimilide concentration-dependently (0.3 – 10 μM) prolonged the action potential duration (APD) in the absence and presence of muscarinic stimulation. In isolated hearts, perfusion of CCh shortened the duration of the monophasic action potential (MAP) and effective refractory period (ERP) of the left atrium and lowered the atrial fibrillation threshold (AFT). Addition of azimilide inhibited the induction of AF by prolonging the duration of MAP and ERP. The I_K.ACh inhibition by azimilide may at least in part contribute to the effectiveness to prevent parasympathetic-type AF.

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The aim of the present study was to elucidate the pathogenic role of endogenous 5-HT in pancreatitis. Injections of cerulein at hourly intervals caused edematous pancreatitis in mice characterized by hyperenzymemia and histological alterations. While the cerulein-induced hyperenzymemia was attenuated in mice pretreated with p-CPA, a 5-HT depletor, it was exaggerated by the preferential 5-HT_2A agonist (DOI), but not by the preferential 5-HT_2B agonist (BW723C86) or the preferential 5-HT_2C agonist (mCPP). Selective 5-HT_2A antagonists (risperidone, spiperone, ketanserin, AMI-193, and MDL 11,939) dose-dependently attenuated the hyperenzymemia; and their potency order, excepting that of ketanserin which has considerable affinity at the 5-HT_2C receptor as well, paralleled their reported pK_i values at the 5-HT_2A receptor. Selective 5-HT_2B (SB204741) and 5-HT_2C (SB242084) antagonists hardly affected the hyperenzymemia. Although the non-selective 5-HT_2A/_2B/_2C antagonists (metergoline, ritanserin, and methysergide) dose-dependently attenuated the hyperenzymemia, they were relatively less potent compared to their high pK_i values at the 5-HT_2A receptor. In another set of experiments, risperidone, but not SB204741 and SB242084, dose-dependently reversed the cerulein-induced histological alteration of the pancreas (inflammatory cell infiltration). These results suggest that endogenously released 5-HT activates 5-HT_2A receptors to aggravate cerulein-induced pancreatitis. We propose that selective 5-HT_2A antagonists may provide a new therapy for acute pancreatitis.

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To elucidate the mechanisms underlying nasal symptoms in patients with aspirin hypersensitivity, we evaluated the effects of orally administered non-steroidal anti-inflammatory drugs (NSAIDs) on the nasal patency of guinea pigs with cedar pollen–induced chronic allergic rhinitis. Indomethacin (10 mg/kg) administered 1 h before a pollen challenge amplified the antigen-induced nasal blockage. More interestingly, even in the absence of the pollen challenge, indomethacin induced nasal blockage at 30 min at 4 h after administration. However, indomethacin-induced nasal blockage was not provoked in non-sensitized animals. Another NSAID, diclofenac (30 mg/kg), also evoked nasal blockage, but unexpectedly, aspirin (500 mg/kg) did not affect nasal patency. Indomethacin-induced nasal blockage was unaffected by a cysteinyl leukotriene receptor (CysLT₁ receptor) antagonist, pranlukast (30 mg/kg, p.o.), or by prostaglandin E₂ (10⁻³ M, intranasal), suggesting that the nasal blockage may not be due to hyperproduction of cysteinyl leukotrienes or inhibition of prostaglandin E₂ production. These results indicate that the indomethacin-induced nasal blockage may not be an identical phenomena to airway symptoms in aspirin hypersensitivity patients. However, because chronic nasal inflammation is indispensable for the development of nasal blockage, indomethacin-induced nasal blockage may become a clue to elucidate new mechanisms underlying hypersensitivity to NSAIDs.

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Phosphoinositide-3 kinase (PI3K) and phospholipase C (PLC) utilize the same phosphoinositides as substrates to produce different signaling molecules. These enzymes are activated by a similar set of cell signaling mechanisms, i.e., tyrosine kinases and G proteins, and affect common cell functions, including proliferation, motility, and intracellular trafficking. Despite these similarities, the interplay between these enzymes is not well understood. To address this issue, the effects of the PI3K inhibitor LY294002 on carbachol-induced calcium increase in PC12h cells were examined. As carbachol stimulates both Gq- and Gi-coupled muscarinic acetylcholine receptors (mAChRs), PI3K and PLC are activated simultaneously in this protocol. LY294002 was found to reduce the carbachol-induced calcium increase, and the reduction was attributed to suppression of calcium entry. As LY294002 did not affect either carbachol-induced calcium release or calcium entry induced by calcium store depletion, this agent was found to suppress calcium entry directly activated by mAChRs. Although PI3K was supposed to compete for substrates with PLC, the PI3K inhibitor did not enhance PLC-dependent cellular responses. As LY294002 was still effective by treating cells after carbachol stimulation, it is likely that this agent blocks the calcium entry channels directly.

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Substance P is known to be released from dorsal root ganglion (DRG) neurons, but the possible involvement of non-neuronal cells in the process of substance P release is not known. During the culture period, the number of surviving DRG neurons, the total substance P content from the culture medium and DRG cells, and the total protein of DRG cells were monitored. Both the number of surviving neurons and the total substance P content decreased in a time-dependent manner, whereas the total protein synthesis was increased. The localization of substance P in small-to-medium–sized neurons was further confirmed by immunocytochemistry. Finally, the substance P release levels from the neuronal and non-neuronal enrichments were investigated by radioimmunoassay. Both partially purified DRG neurons and non-neuronal cells exhibited a weaker substance P release response to capsaicin or KCl, relative to unpurified DRG cells. The total substance P content from the partially purified DRG neurons was almost the same as that from the unpurified DRG cells, but much more than that from the partially purified DRG non-neuronal cells. These findings suggest that substance P is released from DRG neurons, and this process should require the coexistence of neurons and non-neuronal cells.

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Freezing behavior is thought to be a sign of fear in animals. We examined whether the freezing behavior during the elevated open-platform stress, which is a psychological stressor without painful stimulus, is modulated by serotonergic neurotransmission and would be a useful marker for screening anxiolytic and/or antidepressant. Male ICR mice (6 – 8-week-old) were individually placed on an elevated open-platform and the duration of freezing behavior of mouse was measured for 10 min. Fluoxetine and citalopram, selective serotonin (5-HT) reuptake inhibitors, markedly decreased the duration of freezing. Fenfluramine, a 5-HT releaser, and 8-OH-DPAT, a potent 5-HT_1A-receptor agonist, also significantly decreased the duration of freezing. In contrast, the 5-HT-synthesis inhibitor p-chlorophenylalanine significantly increased the duration of freezing. Diazepam, a benzodiazepine anxiolytic, had no effect on the duration of freezing at doses having no effect on locomotor activity. Imipramine and clomipramine, tricyclic antidepressants, also did not affect the duration of freezing. Reboxetine, a selective noradrenaline reuptake inhibitor, significantly increased the duration of freezing. These results indicate that the activation of serotonergic neurotransmission attenuates the fear-related behavior in the elevated open-platform test, while the activation of noradrenergic neurotransmission increases the fear-related behavior. In addition, this test is convenient for assaying anxiolytic drugs that affect serotonergic neurotransmission.

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The responses to adenosine were studied on isolated, methacholine-precontracted tracheal strips of guinea pigs in the course of long-term caffeine or solvent treatment. Guinea pigs were fed caffeine for 10 weeks (average serum caffeine concentration: 39.1 ± 3.9 μM). In epithelium-intact tracheal preparations (EITPs), sensititization to adenosine-induced relaxation (AIR) developed. It attained a maximum in week 1 of caffeine treatment, and then its level diminished and disappeared completely by weeks 4 – 6. In epithelium-denuded tracheal preparations (EDTPs), an increase in the sensitivity to adenosine was observed from week 1 to week 10 (a 4 – 6-fold reduction in EC₅₀). Use of a coaxial bioassay system confirmed the role of epithelium in this process. The enhancement of the AIR of the EITPs was not modified by inhibitors of cyclooxygenase and lipoxygenase. Following depletion of the neuropeptides by acute capsaicin pretreatment, the AIR of the EITPs was strongly enhanced after caffeine treatment for 6 weeks. In chronically caffeine-treated EITPs, the inhibition of neutral endopeptidase led to dramatic reduction of the AIR. On the basis of the results by inhibiting nitric oxide synthase, it can be supposed that nitric oxide released from EITPs of long-lasting caffeine-treated animals operated as a constrictor agent. Our results show that chronic caffeine treatment gives rise to an initial sensitization to adenosine of the EITPs, this being followed by the development of a specific adaptive process in the epithelial cells, which counterbalances the increased tracheal sensitivity to adenosine.

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It has been hypothesized that air pollution has played a role in the increase in allergy prevalence. However, it remains unclear what exact roles are played by polycyclic aromatic hydrocarbons (PAHs), which are encountered in the environment in the form of air pollution, in allergic rhinitis. Thus, we examined whether benzo(a)pyrene (BaP) and 1-nitropyrene (1-NP), representative PAHs, aggravate allergic rhinitis symptoms, using a guinea-pig model. Sensitized animals were repeatedly challenged by inhalation of Japanese cedar pollen once a week. BaP or 1-NP was daily and intranasally administered for 2 weeks (short-term treatment) or for 22 weeks from the time before the sensitization period (long-term treatment). The short-term treatment affected neither nasal blockage nor sneezing induced by antigen. In contrast, the long-term treatment aggravated the antigen-induced nasal blockage that was induced 7 weeks after the start of the treatment with BaP or 1-NP. This aggravation continued during the intranasal treatment with PAH. However, neither sneezing nor Cry j 1-specific IgE antibody production was affected even by the long-term treatment. In conclusion, the long-term treatment with BaP and 1-NP can aggravate allergic rhinitis. The mechanisms underlying this aggravation are not associated with production of Cry j 1-specific IgE.

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