Chikao Shimamoto

Evaluation of Gastric Motor Activity in the Elderly by Electrogastrography and the 13C-Acetate Breath Test

Background: Elderly people frequently have symptoms of fullness and appetite loss due to impaired gastric motor activity. These symptoms may cause malnutrition, immunosuppression and other complications. Objective: The effects of aging and daily activity on gastric motility in the elderly were investigated by electrogastrography and the 13C-acetate breath test. Methods: We enrolled seven active elderly subjects (active elderly group), seven elderly subjects staying at a geriatric facility who had reduced mental and physical capacities (inactive elderly group) and seven healthy young volunteers (young group). Electrogastrography was recorded before and after ingestion of a 13C-acetate-mixed liquid meal. Expired air was sampled every 10 min after the meal to measure the 13CO2 concentration. Results: The ratio of the incidence of the 3-cpm wave (gastric intrinsic frequency) during the postprandial period compared to the fasting state was reduced in both elderly groups compared to young subjects, and the reduction was greater in the inactive elderly than in the active elderly group. The ratio of the amplitude of the peak frequency during the postprandial period to that in the fasting state (power ratio) was also lower in the elderly groups. The time of peak 13CO2 expiration was delayed in the active elderly and more so in the inactive elderly group. Conclusion: Postprandial peristalsis and gastric contractile force are reduced in the elderly, and gastric emptying is delayed indicating a reduction in gastric motor activity.

EP1 and EP4 Receptors Mediate Exocytosis Evoked by Prostaglandin E2 in Guinea‐Pig Antral Mucous Cells

Effects of prostaglandin E2 (PGE2) on exocytosis of mucin were studied in mucous cells isolated from guinea‐pig antrum using video‐microscopy. Stimulation with PGE2 elicited a sustained increase in the frequency of exocytotic events in a dose‐dependent manner, which was under regulation by both Ca2+ and cAMP. Stimulation with a selective prostanoid EP4 receptor agonist (ONO‐AEI‐329, 10 μM), which activates cAMP signals, elicited a sustained increase in the frequency of exocytotic events (30% of that evoked by 1 μM PGE2). Stimulation with an EP1 agonist (17‐P‐T‐PGE2, 1 μM), which activates Ca2+ signals, increased the frequency of exocytotic events to a lesser extent (5% of that evoked by 1 μM PGE2), while addition of an EP1 antagonist (ONO‐8713, 10 μM) decreased the frequency of exocytotic events (approximately 40% of that evoked by 1 μM PGE2). However, addition of the EP1 agonist potentiated the frequency of exocytotic events evoked by the EP4 agonist or forskolin (which elevates cAMP levels) and increased the sensitivity of the exocytotic events to forskolin. These results suggest that the Ca2+ signal activated via the EP1 receptor potentiates the cAMP‐regulated exocytotic events activated via the EP4 receptor during PGE2 stimulation, by increasing the sensitivity of the exocytotic response to cAMP. In conclusion, exocytotic events in PGE2‐stimulated antral mucous cells were regulated by interactions between EP1 and EP4 receptors.

Isosmotic modulation of Ca2+‐regulated exocytosis in guinea‐pig antral mucous cells: role of cell volume

1 Exocytotic events and changes of cell volume in mucous cells from guinea‐pig antrum were examined by video‐enhanced optical microscopy. 2 Acetylcholine (ACh) evoked exocytotic events following cell shrinkage, the frequency and extent of which depended on the ACh concentration. ACh actions were mimicked by ionomycin and thapsigargin, and inhibited by Ca2+‐free solution and Ca2+ channel blockers (Ni2+, Cd2+ and nifedipine). Application of 100 μM W‐7, a calmodulin inhibitor, also inhibited the ACh‐induced exocytotic events. These results indicate that ACh actions are mediated by intracellular Ca2+ concentration ([Ca2+]i) in antral mucous cells. 3 The effects of ion channel blockers on exocytotic events and cell shrinkage evoked by ACh were examined. Inhibition of KCl release (quinine, Ba2+, NPPB or KCl solution) suppressed both the exocytotic events and cell shrinkage evoked by ACh. 4 Bumetanide (inhibition of NaCl entry) or Cl−‐free solution (increasing Cl− release and inhibition of NaCl entry) evoked exocytotic events following cell shrinkage in unstimulated antral mucous cells and caused further cell shrinkage and increases in the frequency of exocytotic events in ACh‐stimulated cells. However, Cl−‐free solution did not evoke exocytotic events in unstimulated cells in the absence of extracellular Ca2+, although cell shrinkage occurred. 5 To examine the effects of cell volume on ACh‐evoked exocytosis, the cell volume was altered by increasing the extracellular K+ concentration. The results showed that cell shrinkage increases the frequency of ACh‐evoked exocytotic events and cell swelling decreases them. 6 Osmotic shrinkage or swelling caused the frequency of ACh‐evoked exocytotic events to increase. This suggests that the effects of cell volume on ACh‐evoked exocytosis under anisosmotic conditions may not be the same as those under isosmotic conditions. 7 In antral mucous cells, Ca2+‐regulated exocytosis is modulated by cell shrinkage under isosmotic conditions.

Ciliary beat frequency controlled by oestradiol and progesterone during ovarian cycle in guinea‐pig Fallopian tube

The ciliary beat frequency (CBF) of guinea‐pig fimbria during the ovarian cycle was measured by video microscopy using a high‐speed camera (500 Hz). In the follicular phase, with increasing concentrations of β‐oestradiol ([βE2]) and a low concentration of progesterone ([PRG]), CBF increased from 13.5 to 16 Hz. In the ovulatory phase, with further increase of [βE2], CBF decreased gradually from 16 to 13.5 Hz. In the early luteal phase, with low [PRG] and [βE2], CBF increased to ∼17 Hz; however, in the middle luteal phase, with increasing [PRG], CBF decreased (∼12 Hz), and in the late luteal phase, with decreasing [PRG], CBF increased to ∼15 Hz. Then, in the resting phase, with low [βE2] and [PRG], CBF decreased immediately to ∼14 Hz. The CBF of the fimbria was measured in guinea‐pigs treated with β‐oestradiol benzoate (βE2B) or medroxyprogesterone (mPRG). A low dose of βE2B increased CBF to ∼14.5 Hz, whereas a high dose decreased it to ∼11 Hz. A βE2 receptor blocker, ICI‐182,780, abolished the βE2B‐induced CBF changes and maintained CBF at ∼12.0 Hz. Medroxyprogesterone decreased CBF to ∼12.5 Hz, and mifepristone (a PRG receptor blocker) abolished the mPRG‐induced CBF decrease and maintained CBF at ∼15 Hz. The addition of both blockers increased CBF to ∼18 Hz, suggesting that activation of βE2 or PRG receptors decreases the CBF of the fimbria. In conclusion, a moderate [βE2] increase maintains a high CBF (15.5 Hz) in the follicular phase, and then further [βE2] increase decreases CBF to 13.5 Hz in the ovulatory phase. In the early and late luteal phase, low [βE2] and [PRG] increase CBF to 17 and 15 Hz, respectively, and in the middle luteal phase a high [PRG] decreases CBF (to ∼12 Hz). Thus, the CBF of the fimbria was controlled by signals via βE2 and PRG receptors in guinea‐pigs.

Hypo‐osmotic potentiation of acetylcholine‐stimulated ciliary beat frequency through ATP release in rat tracheal ciliary cells

The ciliary beat frequency (CBF) of rat tracheal ciliary cells in a slice preparation was measured using video‐enhanced contrast (VEC) microscopy. Acetylcholine (ACh) increased CBF mediated via intracellular Ca2+ concentration ([Ca2+]i) in a dose‐dependent manner. An adequate hypo‐osmotic stress (−40 mosm) potentiated ACh‐stimulated CBF increase in tracheal ciliary cells and shifted the ACh dose–response curve to the left (lower concentration side). This potentiation was independent of hypo‐osmotic stresses applied ranging from −20 mosM to −90 mosM. A hypo‐osmotic stress induces ATP release in many cell types. The present study demonstrated that suramin (an inhibitor of purinergic receptors) and apyrase (an ATPase/ADPase) eliminate the hypo‐osmotic potentiation of ACh‐stimulated CBF increase and that ATP increased [Ca2+]i and CBF, as well as potentiating ACh‐stimulated rises in [Ca2+]i and CBF increase. Moreover, the apical surface of tracheal ciliary cells were stained immunopositive for the P2X4 purinergic receptor. A hypo‐osmotic stress (−40 mosM) transiently increased [Ca2+]i and potentiated the ACh‐stimulated [Ca2+]i increase. The hypo‐osmotic potentiation of ACh‐stimulated CBF increase was not detected under Ca2+‐free conditions. These observations suggest that a hypo‐osmotic stress stimulates ATP release from the trachea. The released ATP may induce further increases in [Ca2+]i and CBF in ACh‐stimulated tracheal ciliary cells, which may be mediated by purinergic receptors, such as P2X4.

Alteration of colonic mucin after ureterosigmoidostomy.

PURPOSE: Patients undergoing urinary diversion by ureterosigmoidostomy after complete cystectomy for malignant bladder tumors show a high incidence of neoplasia at and near the site of anastomosis. We examined a risk factor for tumor occurrence in the area of anastomosis, alterations of mucus glycoproteins in the surrounding colonic mucosa. METHODS: Colonoscopy was performed in 37 patients who had undergone ureterosigmoidostomy. Biopsy specimens were obtained near the ureteral anastomosis and were stained with hematoxylin and eosin, high iron-diamine alcian blue (pH 2.5), and a fluorescent lectin conjugate (peanut agglutinin). RESULTS: At the anastomotic site colonoscopy showed protruding lesions in 26 of 37 patients (71 percent), all histologically representing inflammatory granulomas. The mucosa around the anastomosis was normal in endoscopic appearance; however, histologically, slight inflammatory cell infiltration, edema, and increased numbers of Paneth cells were observed. Alcian blue staining revealed an increase in mucosal sialomucin postoperatively compared with preoperatively. The proportion of peanut agglutinin-binding mucin, not observed in normal mucosa but seen in malignant or premalignant tissue, was increased. CONCLUSION: As postoperative interval increases, changes in properties of the “background” mucosa become greater, which suggests an association with colonic carcinogenesis.

Prostaglandin E2 release in gastric antral mucosa of guinea‐pigs: basal PGE2 release by cyclo‐oxygenase 2 and ACh‐stimulated PGE2 release by cyclo‐oxygenase 1

Prostaglandin E2 (PGE2), which is generated by two isoforms of cyclo‐oxygenase (COX1 and COX2), is a key mediator in gastric mucosal defense. In the present study, antral mucosa of guinea‐pigs was incubated with various agonists or antagonists in a medium, the PGE2 concentration of which was measured using a PGE2 EIA kit. Prostaglandin E2 was released from the antral mucosa spontaneously (basal PGE2 release) and acetylcholine (ACh, 10 μm) enhanced the PGE2 release (ACh‐stimulated PGE2 release) was mediated via intracellular Ca2+ concentration ([Ca2+]i). Arachidonic acid enhanced both forms of PGE2 release, and a phospholipase A2 inhibitor (amylcinnamoyl anthranilic acid) and COX inhibitors (acetylsalicylic acid and indomethacin) decreased them. 5‐(4‐Chlorophenyl)‐1‐(4‐methoxyphenyl)‐3‐trifluoromethylpyrazol (SC560, 100 nm, a COX1‐selective inhibitor) inhibited ACh‐stimulated PGE2 release without any decrease in basal PGE2 release. N‐(2‐Cyclohexyloxy‐4‐nitrophenyl) methanesulphonamide (NS398, 20 μm, a COX2‐selective inhibitor) decreased basal PGE2 release without any reduction of ACh‐stimulated PGE2 release. However, ionomycin (a Ca2+ ionophore) increased PGE2 release from antral mucosa in the presence of SC560 or NS398, suggesting that COX1 and COX2 are regulated by [Ca2+]i. These findings indicate that COX1‐containing cells have ACh receptors but COX2‐containing cells do not. Moreover, in isolated antral epithelial cells, SC560 decreased basal and ACh‐stimulated PGE2 release, but NS398 did not. In conclusion, in antral mucosa, basal PGE2 release is mainly maintained by COX2 of non‐epithelial cells, and ACh‐stimulated PGE2 release is maintained by COX1 of epithelial cells.

Enhancement of Ca2+‐regulated exocytosis by indomethacin in guinea‐pig antral mucous cells: arachidonic acid accumulation

Ca2+‐regulated exocytosis is enhanced by an autocrine mechanism via the PGE2–cAMP pathway in antral mucous cells of guinea‐pigs. The inhibition of the PGE2–cAMP pathway by H‐89 (an inhibitor of protein kinase A, PKA) or aspirin (ASA, an inhibitor of cyclo‐oxygenase, COX) decreased the frequency of ACh‐stimulated exocytotic events by 60%. Indomethacin (IDM, an inhibitor of COX), however, decreased the frequency of ACh‐stimulated exocytotic events only by 30%. Moreover, IDM increased the frequency of ACh‐stimulated exocytotic events by 50% in H‐89‐treated or ASA‐treated cells. IDM inhibits the synthesis of Prostaglandin (PGG/H) and (15R)‐15‐hydroxy‐5,8,11 cis‐13‐trans‐eicosatetraenoic acid (15R‐HPETE), while ASA inhibits only the synthesis of PGG/H. Thus, IDM may accumulate arachidonic acid (AA). AACOCF3 or N‐(p‐amylcinnamoyl) anthranilic acid (ACA; both inhibitors of phospholipase A2, PLA2), which inhibits AA synthesis, decreased the frequency of ACh‐stimulated exocytotic events by 60%. IDM, however, did not increase the frequency in AACOCF3‐treated cells. AA increased the frequency of ACh‐stimulated exocytotic events in AACOCF3‐ or ASA‐treated cells, similar to IDM in ASA‐ and H‐89‐treated cells. Moreover, in the presence of AA, IDM did not increase the frequency of ACh‐stimulated exocytotic events in ASA‐treated cells. The PGE2 release from antral mucosa indicates that inhibition of PLA2 by ACA inhibits the AA accumulation in unstimulated and ACh‐stimulated antral mucosa. The dose–response study of AA and IDM demonstrated that the concentration of intracellular AA accumulated by IDM is less than 100 nm. In conclusion, IDM modulates the ACh‐stimulated exocytosis via AA accumulation in antral mucous cells.

Delayed shrinkage triggered by the Na+–K+ pump in terbutaline‐stimulated rat alveolar type II cells

Terbutaline (10 μm) induced a triphasic volume change in alveolar type II (AT‐II) cells: an initial shrinkage (initial phase) followed by cell swelling (second phase) and a gradual shrinkage (third phase). The present study demonstrated that the initial and the third phases are evoked by the activation of K+ and Cl− channels and the second phase is evoked by the activation of Na+ and Cl− channels. Ouabain blocked the third phase, although it did not block the initial and second phases. This suggests that the third phase is triggered by the Na+–K+ pump. Tetraethylammonium (TEA, a K+ channel blocker) decreased the volume of AT‐II cells and enhanced the terbutaline‐stimulated third phase, although quinidine, another K+ channel blocker, increased the volume of AT‐II cells. The TEA‐induced cell shrinkage was inhibited by ouabain, suggesting that TEA increases Na+–K+ pump activity. Ba2+, 2,3‐diaminopyridine and a high [K+]o (30 mm) similarly decreased the volume of AT‐II cells. These findings suggest that depolarization induced by TEA increases Na+–K+ pump activity, which increases [K+]i. This [K+]i increase, in turn, hyperpolarizes membrane potential. Valinomycin (a K+ ionophore), which induces hyperpolarization, decreased the volume of AT‐II cells and enhanced the third phase in these cells. In conclusion, in terbutaline‐stimulated AT‐II cells, an increase in Na+–K+ pump activity hyperpolarizes the membrane potential and triggers the third phase by switching net ion transport from NaCl entry to KCl release.

Procaterol-stimulated Increases in Ciliary Bend Amplitude and Ciliary Beat Frequency in Mouse Bronchioles

The beating cilia play a key role in lung mucociliary transport. The ciliary beating frequency (CBF) and ciliary bend amplitude (CBA) of isolated mouse bronchiolar ciliary cells were measured using a light microscope equipped with a high-speed camera (500 Hz). Procaterol (aβ 2-agonist) increased CBA and CBF in a dose dependent manner via cAMP. The time course of CBA increase is distinct from that of CBF increase: procaterol at 10 nM first increased CBA and then CBF. Moreover, 10 pM procaterol increased CBA, not CBF, whereas 10 nM procaterol increased both CBA and CBF. Concentration-response studies of procaterol demonstrated that the CBA curve was shifted to a lower concentration than the CBF curve, which suggests that CBA regulation is different from CBF regulation. Measurements of microbead movements on the bronchiole of lung slices revealed that 10 pM procaterol increased the rate of ciliary transport by 37% and 10 nM procaterol increased it by 70%. In conclusion, we have shown that increased CBA is of particular importance for increasing the bronchiolar ciliary transport rate, although CBF also plays a role in increasing it.