Yoh-ichi Satoh

Acetylcholine-induced calcium signaling along the rat colonic crypt axis

BACKGROUND & AIMS Acetylcholine-induced calcium signaling dynamics have been described in cell monolayers derived from colonic mucosa, but not in intact colonic crypts. The aim of this study was to characterize the spatiotemporal characteristics of calcium signaling induced by acetylcholine in isolated intact rat colonic crypts and to identify the muscarinic receptor subtype coupled to this signaling pathway. METHODS Isolated crypts from the distal colon of male Wistar rats were loaded with the calcium-sensitive dye Fura-2 and imaged with a charge-coupled device video camera. RESULTS Acetylcholine mobilized intracellular calcium with an EC50 of 3.9 micromol/L. The response was initiated at the base of the crypt and progressed toward the surface. The velocity of propagation was dose dependent. Addition of muscarinic antagonists inhibited the response (pKb values calculated for pirenzepine and 4-DAMP, 6.08 and 8.65, respectively). Microperfusion of acetylcholine initiated a calcium signal throughout the lower half of the crypt. Microinjection of inositol 1,4,5-triphosphate induced a propagation of a calcium signal along the crypt axis. Heptanol inhibited the velocity of acetylcholine-induced wave propagation by 33%. CONCLUSIONS M3 muscarinic receptors are coupled to the mobilization of calcium from intracellular stores of intact, isolated rat colonic crypts. Intercellular communication potentiates the propagation of the acetylcholine-induced calcium signal along the crypt axis.

Effects of carbachol and catecholamines on ultrastructure and intracellular calcium-ion dynamics of acinar and myoepithelial cells of lacrimal glands.

Abstract.The current study was carried out to investigate autonomic nervous control of secretory functions in the lacrimal gland. To distinguish the difference between the responses to cholinergic and adrenergic agonists in acinar and myoepithelial cells in the lacrimal gland of guinea pigs, the morphological and functional responses to the agonists were examined by electron microscopy and by digital-imaging analysis of the intracellular concentration of Ca2+ ([Ca2+]i) using fluorescent Ca2+-indicators (Fura-2/AM and Indo-1/AM). In the resting state, exocytosis was rare, and the [Ca2+]i in acinar and myoepithelial cells was low (less than 300 nM). Stimulation with carbachol (CCh) induced a rapid rise in [Ca2+]i reaching a peak level followed by gradual decay and an appearance of many exocytotic figures. Approximately 4–8 s after an initial increase of [Ca2+]i, myoepithelial cells commenced contraction. Noradrenaline or adrenaline induced an increase in [Ca2+]i and exocytosis in acinar cells, but caused no [Ca2+]i increase in myoepithelial cells. In a Ca2+-deficient environment, the responses to CCh in myoepithelial cells and those to noradrenaline in acinar cells were inhibited, whereas the responses to CCh in acinar cells remained unchanged. Isoproterenol caused no effect on [Ca2+]i dynamics, although it occasionally induced exocytosis. Different cellular signaling pathways may be involved in the responses in acinar and in myoepithelial cells to different agonists. Lacrimation mechanisms are redundant.

Immunohistochemical observations of lysozyme in the Paneth cells of specific-pathogen-free and germ-free mice.

The localization of lysozyme, which may function as an antibacterial agent, was immunohistochemically studied on the mouse Paneth cell secretory granules showing bipartite substructures (central core and peripheral halo). The lysozyme immunoreactivity was observed in the core, but not in the halo. Even in germ-free mice, Paneth cells have many secretory granules and their cores show lysozyme immunoreactivity. It seems likely that mice Paneth cells possess the ability to produce secretory granules containing lysozyme in disregard of the intestinal bacterial milieu.

Secretagogue-induced apocrine secretion in the Harderian gland of the rat

Abstract.Harderian glands of male albino rats were stimulated with secretagogues and examined by transmission and scanning electron microscopy for the purpose of studying the apocrine secretory mechanism. Rats in the control group were perfused with standard HEPES-buffered Ringer’s solution. Their glandular endpieces showed wide lumina that contained few secretory materials; spontaneous exocytosis was sometimes observed. However, there were no features suggestive of an apocrine secretory mechanism or myoepithelial cell contractions. After stimulation with NaF+AlCl3 or carbachol in HEPES-buffered Ringer’s solution, the rats shed ”bloody tears” and the glandular lumina were jammed with apical protrusions, cytoplasmic material and secretory products. The basal surface of the glandular cells showed bulging caused by myoepithelial cell contraction. Perfusion with HEPES-buffered Ringer’s solution containing papaverine inhibited secretagogue-induced myoepithelial cell contraction but not the enhanced secretory activities of the glandular cells. The present results demonstrate that the Harderian gland of the rat can release secretory material not only by exocytosis, but also by an apocrine mechanism under stimulating conditions, and that myoepithelial cell contraction may not be involved in causing apical protrusion in the glandular cells.

Carbamylcholine-induced morphological changes and spatial dynamics of [Ca2+]c in Harderian glands of guinea pigs: calcium-dependent lipid secretion and contraction of myoepithelial cells

To determine whether lipid-secreting cells have cytosolic Ca2+ concentration ([Ca2+]c)-related secretory mechanisms, morphological changes and intracellular calcium dynamics of Harderian glands of guinea pigs stimulated by secretagogs were studied by electron microspy and Fura-2/AM digital image analysis. Control glandular cells contained large lipid vacuoles that were bordered by multi-layered membranes. Rough-surfaced endoplasmic reticulum, mitochondria, and smooth-surfaced endoplasmic reticulum may be involved in lipid vacuole formation. Myoepithelial cells surrounded alveoli. After carbamylcholine (CCh, 10−6, 10−5, and 10−3 M) stimulation, lipid materials within the membranous structures were frequently discharged by an exocytotic mechanism. Conspicuous deformation of glandular cells caused by vigorous contraction of myoepithelial cells was observed in isolated alveoli after 10−6M CCh stimulation, whereas the deformaties of glandular tissues perfused via vessels were small even after 10−3M CCh stimulation. Connective tissue between glandular alveoli inhibited unbridled myoepithelial-cell contraction. Fura-2/AM digital imaging analysis revealed that CCh stimulation caused an increase in [Ca2+]c in isolated alveoli. The morphological reactions and changes in [Ca2+]c were prevented by atropine. When extracellular calcium ions were absent, enhanced extrusion of lipid vacuoles, myoepithelial-cell contraction, and a rise in [Ca2+]c after CCh stimulation were not observed. Nicotine and catecholamines had no effect on the secretion or on the dynamics of [Ca2+]c. It can be concluded that acetylcholine elicits exocytosis in glandular cells and contraction of the myoepithelial cells of Harderian glands, accompanied by an increase in [Ca2+]c. The dynamics of [Ca2+]c of the gland alveoli are mostly dependent on extracellular Ca2+.

Effects of AlF4– and ATP on intracellular calcium dynamics of crypt epithelial cells in mouse small intestine

Abstract. Previous digital imaging analysis of intracellular calcium ion ([Ca2+]i) dynamics in the crypt of the small intestine showed little response by most columnar cells to cholinergic and adrenergic agonists. The objective of the present study was to demonstrate whether G-protein activators and other transmitters elicit [Ca2+]i changes in crypt cells. We used digital imaging to analyze spatiotemporal dynamics of [Ca2+]i in Fura-2/AM-loaded isolated crypts of mouse duodenum and ileum. AlF4– increased [Ca2+]i in crypt columnar cells. In many cases, we observed [Ca2+]i oscillations, which were synchronized throughout the entire crypt. The oscillations were blocked by octanol. ATP, but not adenosine, caused a [Ca2+]i increase in middle crypt-regions of the duodenum and upper regions of the ileum, and the [Ca2+]i wave propagated towards the crypt bottom. The ATP-induced [Ca2+]i increase was prevented by pretreatment with thapsigargin or suramin, but not by La3+ or an extracellular Ca2+-free environment. Neither dopamine, 5-hydroxytryptamine (5-HT), histamine, vasoactive intestinal peptide, substance P, cholera toxin, nor guanylin had significant effects. The [Ca2+]i dynamics of Paneth cells were independent of the AlF4–-induced synchronous oscillations of columnar cells and of the ATP-induced [Ca2+]i wave. In conclusion, crypt columnar cells have [Ca2+]i-dependent intracellular signaling mechanisms that are linked with G proteins, and by which the cells communicate with each other. ATP elicited [Ca2+]i mobilization from columnar cells via P2 receptors, although some regional differences were noted between the duodenum and ileum.

Lipid secretory mechanisms in the mammalian Harderian gland

The mammalian Harderian glands are lipid‐secreting glands. In an unstimulated condition, the glandular cells frequently exocytose the lipid materials; however, no intracellular calcium ion ([Ca2+]c) changes are detectable. Cholinergic (muscarinic) secretagogues induce secretory activity and increase of [Ca2+]c. A G‐protein activator, sodium fluoride, enhances the secretory activity and increase of [Ca2+]c. Removal of extracellular calcium ions inhibits the secretion enhanced by cholinergic stimulation. Under pharmacologic stimulation, glandular cells may show an apocrine‐like secretory pattern. Cholinergic stimulation also induces contraction of the myoepithelial cells covering glandular end pieces; however, the reduction in volume of glandular end pieces is not prominent. Catecholamines have no effect on the release of lipid materials. These results indicate the involvement of G‐proteins linking with muscarinic receptors and Ca2+ dynamics (increase of [Ca2+]c and Ca2+ influx) in lipid secretion by glandular cells and in contraction of myoepithelial cells of mammalian Harderian glands. However, the increase of [Ca2+]c in Harderian glands was less when compared with other cells—for instance, those which secrete protein.

Mast cell degranulation is negatively regulated by the Munc13-4-binding small-guanosine triphosphatase Rab37.

Mast cell degranulation is regulated by the small guanosine triphosphatases (GTPases) Rab27a and Rab27b, which have distinct and opposing roles: Rab27b acts as a positive regulator through its effector protein Munc13-4, a non-neuronal isoform of the vesicle-priming Munc13 family of proteins, whereas Rab27a acts as a negative regulator through its effector protein melanophilin, by maintaining integrity of cortical filamentous actin (F-actin), a barrier to degranulation. Here we investigated the role of Rab37, one of the Rab GTPases assumed to be implicated in regulated secretion during mast cell degranulation. Using the RBL-2H3 mast cell line, we detected Rab37 on the secretory granules and found that antigen-induced degranulation was extensively increased by either knockdown of Rab37 or overexpression of a dominant-active Rab37 mutant. This hypersecretion phenotype in the Rab37-knockdown cells was suppressed by simultaneous knockdown of Rab27a and Rab27b or of Munc13-4, but not by disruption of cortical F-actin. We further found that Rab37 interacted with Munc13-4 in a GTP-independent manner and formed a Rab27-Munc13-4-Rab37 complex. These results suggest that Rab37 is a Munc13-4-binding protein that inhibits mast cell degranulation through its effector protein, by counteracting the vesicle-priming activity of the Rab27-Munc13-4 system.

Spontaneous [Ca2+]i oscillations in G1/S phase-synchronized cells.

Ca(2+) signaling controls a wide range of cellular functions such as division, fertilization, apoptosis and necrosis. Specifically, calcium signaling is thought to play a crucial role in driving cells through the different stages of the cell-division cycle. In most cells, however, this fact is far from being established. Few studies have examined this question from a different perspective: whether cells exhibit some characteristic cell cycle-dependent intracellular calcium-signaling patterns. This approach is effective in discerning the causal relationship between Ca(2+) signaling and the cell cycle. Through synchronization of the cell cycle, flow cytometry and confocal scanning microscopic intracellular calcium ion concentration ([Ca(2+)](i)) imaging, the present study shows that the G1/S phase transition is uniquely characterized by spontaneous [Ca(2+)](i) oscillations that last for up to 40 min. Most likely, these oscillations emanate from the [Ca(2+)](i) signaling that accompanies DNA replication as the cell prepares for the next division cycle. These temporal signals further affirm the significance of Ca(2+) in the cell cycle.

α1-Adrenoceptors relate Ca 2+ modulation and protein secretions in rat lacrimal gland

Noradrenaline (NA) is a catecholamine with multiple roles including as a hormone and a neurotransmitter. Cellular secretory activities are enhanced by adrenergic stimuli as well as by cholinergic stimuli. The present study aimed to determine which adrenoceptors play a role in controlling intracellular calcium ion ([Ca(2+)]i) level in acinar cells of rat lacrimal glands. Expression of mRNA for adrenoceptor subtypes in the acinar cells was assessed using RT-PCR. All types except α2c, β1, and β3 were detected. NA induced a [Ca(2+)]i increase with a biphasic pattern in the acinar cells. Removal of extracellular Ca(2+) and use of Ca(2+)-channel blockers did not inhibit the NA-induced [Ca(2+)]i increases. In contrast, U73122 and suramin almost blocked these increases. The α1-adrenoceptor agonist phenylephrine induced a strong increase in [Ca(2+)]i. However, clonidine and isoproterenol failed to induce a [Ca(2+)]i increase. The peroxidase activity was quantified as a measure of mucin secretion. Ca(2+)-dependent exocytotic secretion of peroxidase was detected in rat lacrimal glands. The RT-PCR results showed that MUC1, MUC4, MUC5AC, MUC5B, and MUC16 were expressed in acinar cells. These findings indicated that NA activates α1-adrenoceptors, which were found to be the main receptors in Ca(2+)-related cell homeostasis and protein (including mucin) secretion in lacrimal glands.