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We investigated the existence of chromogranin A (CgA) in salivary glands of the horse by Western blotting and enzyme immunoassay (EIA) using an antiserum against a peptide sequence of equine CgA. We also compared its cellular distribution between the horse and rat salivary glands with a tyramide signal amplification immunofluorescence technique. Western blotting gave three significant immunoreactive bands (74, 56 and 48 kDa) in adrenal medulla and three major salivary glands of horses. Immunoreactivities for CgA measured by EIA in horses were 154.05 ± 41.46, 20.32 ± 5.59 and 4.43 ± 2.23 pmol/g wet weight in the parotid gland, submandibular gland and sublingual gland, respectively, and 1.03 ± 0.407 pmol/mg protein in the saliva. Immunohistochemically, the positive reactivity was mainly recognized at acinar cells in equine salivary glands. This exhibits a contrast to the finding in the rat salivary glands that the CgA immunoreactivity is localized at the duct cells of the submandibular gland. These results provide novel evidence that in the horse, CgA is stored in the acinar cells of salivary glands, and secreted into saliva.

Double immunohistochemical staining detected inositol 1, 4, 5-trisphosphate receptor type 2 immunoreactivity (IP 3 R2 IR) and chromogranin A-like immunoreactivity (CgA-like IR) on the same sections prepared from the isolated and perfused submandibular gland of rat. In the control sections prepared from resting state, an intense IP 3 R2 IR was localized at the apical pole of granulated duct cell containing CgA-like IR. Electron-microscopically, the granular cells in the resting state stored numerous membrane-bound granules in the apical cytoplasm. The combined stimulation with 1 μM noradrenaline and 0.1 μM acetylcholine caused immediate maximum increase in secretory response (CgA-like IR secretion, flow, and protein secretion). When the sections were prepared from the gland at the peak of secretory response, the apically converged IP 3 R2 IR became diffuse and indistinguishable. Ultrastructure of the maximally stimulated cells exhibited extensive compound exocytosis in the apical half of the granular duct cells. The secretory responses to the combined stimulation were significantly inhibited by 100 μM 2-aminoethyl diphenylborinate, an inhibitory modulator of IP 3 -mediated Ca 2 + release from intracellular stores, and the intense IP 3 R2 IR was well preserved in the apical pole of granular duct cells containing CgA-like IR. These results may be compatible with the view that the divergence of apically convergent IP 3 R2 with the progress of compound exocytosis may result in decay of [Ca 2 + ], due to divergence of IP 3 R2-sensitive Ca 2 + pool, and this may provide a novel paradigm for desensitization of G protein-coupled signaling system.

To elucidate the detailed process of exocytosis at the highest possible accuracy, we dissociated the pancreatic acinus of the guinea pig and observed zymogen granules under a video-enhanced contrast differential interference contrast (VEC-DIC) microscope. The preparation was thin enough to resolve each zymogen granule with the best clarity. When acinar cells were stimulated with ACh (20 μM), many zymogen granules near the lumen showed an abrupt light intensity change. For a period of 10 s immediately before exocytosis, zymogen granules neither shifted their position nor altered their shape within an accuracy of 38 nm. The time required for individual granules to change the light intensity (the releasing time) ranged from 0.15 to 0.70 s. After each response, the granule maintained its altered contrast for a few seconds until it was retrieved to a planar membrane. No compound exocytosis including granule-granule fusion was observed. We concluded that the exocytosis is not directly initiated by any supramolecular change but by a purely molecular event.

Chromogranin A (CGA) is a member of a family of highly acidic proteins co-stored and co-released with catecholamines in the adrenal medullary cells as well as in other neurons and paraneurons. The nucleotide sequence encoding equine CGA was determined using RT-PCR and rapid amplification of complementary DNA (cDNA) ends (RACE) techniques. A total 1,828 bp of the nucleotide sequence reveals that equine CGA is a 448-residue protein preceded by an 18-residue signal peptide. Comparison of the amino acid sequence of equine CGA with those of human, porcine, bovine, mouse, rat and frog CGA showed high conservation at the NH2-terminal 1-77 amino acids regions (94.8%, 93.5%, 92.2%, 81.8%, 83.1% and 66.2%, respectively) and COOH-terminal 314-430 amino acids regions (90.6%, 81.4%, 90.6%, 80.5%, 83.3% and 39.0%, respectively), as well as a potential dibasic cleavage site, whereas the middle portion showed marked sequence variation (52.5%, 49.1%, 38.9%, 26.6%, 27.9% and 6.2%, respectively). Northern blot analysis and RT-PCR elucidated the tissue distribution of equine CGA mRNA. Its expression was confirmed not only in the adrenal medullary cells but also in other organs (cerebrum, cerebellum, pituitary gland, spinal cord, liver, thyroid gland, striated muscle, lung, spleen, kidney, parotid gland and sublingual gland). Further, in adrenal chromaffin cells and pituitary cells of the anterior-intermediate lobe, the expression was confirmed by in situ hybridization with anti-sense CGA cRNA probe.

Ratiometric images of cytoplasmic Ca2+ concentration ([Ca2+]c) in individual cells were recorded simultaneously with a confocal ultraviolet-laser microscope in the Indo-1-loaded islets isolated from mice. After changes in [Ca2+]c in response to glucose or amino acids were recorded, the islet was fixed, permeabilized, and stained by the indirect immunofluorescence method against insulin or glucagon in situ; the individual cells were then identified in the focal plain identical to that used for the [Ca2+]c imaging. Almost all cells identified as insulin-positive (beta-cells) by their distinct immunofluorescence responded to the increase in glucose concentration from 3 to 11 mmol/l with an increase in [Ca2+]c. Major populations of cells (approximately 65%) identified as glucagon-positive (alpha-cells) responded to the addition of arginine (5-10 mmol/l) to 3 mmol/l glucose solution with an increase in [Ca2+]c. About half of the alpha-cells (47.6%) responded to the addition of alanine (5-10 mmol/l) to 3 mmol/l glucose solution with an increase in [Ca2+]c. In contrast

A large fraction of rat adrenal chromaffin cells (about 60%) shows spontaneous [Ca2+]c oscillations and spontaneous action potentials. In the present study the effects of gamma-aminobutyric acid (GABA) on the spontaneous [Ca2+]c oscillations and electrical properties of rat adrenal chromaffin cells were investigated using Fura-2 [Ca2+]c imaging and patch clamp techniques. GABA inhibited the spontaneous [Ca2+]c oscillations in a reversible manner. The effect of GABA was mimicked by the GABAA and GABAC receptor agonist, muscimol, but not by the GABAB receptor agonist, baclofen. Moreover, the effect was antagonized by the selective GABAA receptor antagonist, bicuculline. The mode of the inhibition was all-or-none, and the threshold concentration at which the inhibition occurred varied widely (50 microM to over 1 microM) from cell to cell. GABA (100 microM) elicited a transient burst of action potentials of diminished amplitude, which was followed by arrest of action potentials. Further analysis showed that GABA (100 microM) induced inward whole-cell currents in voltage-clamp experiments and produced depolarization and membrane conductance increase in current-clamp experiments. The effects appear to be due to an increase in chloride ion conductance since the degree of GABA-induced depolarization depended on the pipette [Cl-]. These results suggest that GABA, acting through GABAA receptor, may play a role in the physiological regulation of rat adrenal chromaffin cells by directly modifying the discharge of spontaneous action potentials and spontaneous [Ca2+]c oscillations.

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. © 1996 Wiley-Liss, Inc.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been reported to increase intracellular Ca2+ concentrations ([Ca2+]i) and catecholamine release in adrenal chromaffin cells. We measured [Ca2+]i with fura-2 and recorded ion currents and membrane potentials with the whole cell configuration of the patch-clamp technique to elucidate the mechanism of PACAP-induced [Ca2+]i increase in bovine adrenal chromaffin cells. PACAP caused [Ca2+]i to increase due to Ca2+ release and Ca2+ influx, and this was accompanied by membrane depolarization and inward currents. The Ca2+ release was suppressed by ryanodine, an inhibitor of caffeine-sensitive Ca2+ stores, but was unaffected by cinnarizine, an inhibitor of inositol trisphosphate-induced Ca2+ release. Ca2+ influx and inward currents were both inhibited by replacement of extracellular Na+, and Ca2+ influx was inhibited by nicardipine, an L-type Ca2+ channel blocker, or by staurosporine, a protein kinase C (PKC) inhibitor, but was unaffected by a combination of omega- conotoxin-GVIA, omega-agatoxin-IVA, and omega-conotoxin- MVIIC, blockers of N-, P-, and Q-type Ca2+ channels. Moreover, 1-oleoyl-2-acetyl-sn-glycerol, a PKC activator, induced inward currents and Ca2+ influx. These results indicate that PACAP causes both Ca2+ release, mainly from caffeine-sensitive Ca2+ stores, and Ca2+ influx via L-type Ca2+ channels activated by membrane depolarization that depends on PKC-mediated Na+ influx.

Cooling is known to inhibit glucose-induced insulin secretion from pancreatic islets, but temperature-dependent processes in stimulus-secretion coupling remain unclear. In the present study, we examin

The effects of lowering extracellular Na+ concentration [Na+]o, on cytosolic Ca2+ concentration, [Ca2+]c were examined by a microfluorimetric method using fura-2 in perifused preparations of isolated rat pancreatic islets. The total replacement of extracellular Na+ (Na+o) by equimolar N-methyl-D-(--)-glucamine caused a rapid rise in [Ca2+]c, and partial replacement of Na+o resulted in correlative rises in [Ca2+]c in accordance with the magnitude of reduced [Na+]o. The rise in [Ca2+]c induced by Na+o removal was strongly inhibited in the Ca2+o-deficient environment or by Ni2+. The [Ca2+]c rise, however, remained almost unchanged in the presence of nifedipine or SK&F 96365, and was enhanced by the addition of ouabain. The electrochemical gradients for Ca2+ (delta mu Ca2+) and Na+ (delta mu Na+) were calculated to be 39.08 and 12.8 kJ/mol, respectively, in this study, indicating a stoichiometry of 3Na+: 1 Ca2+. These results indicate that, in rat pancreatic islets, the rise in [Ca2+]c induced by lowering [Na+]o is mainly due to Ca2+ entry medicated by the Na+/Ca2+ exchanger operating with the stoichiometry of 3Na+:1 Ca2+, and that the Na+/Ca2+ exchanger plays an important role in maintaining stable-state [Ca2+]c.

In order to approach impaired stimulus-secretion coupling in acute pancreatitis induced by a choline-deficient, ethionine-supplemented (CDE) diet in mice, the agonist-evoked intracellular Ca2+

The mechanism of the potentiating effect of vasoactive intestinal peptide (VIP) on cholecystokinin (CCK-8)-induced amylase release was studied in isolated and perifused pancreatic acini of the guinea pig. VIP (30 pM-10 nM) potentiated CCK-8 (100 pM)-induced amylase release. Unexpectedly, VIP inhibited CCK-8-induced intracellular Ca2+ oscillations. Forskolin (10 microM), an activator of adenylate cyclase, potentiated CCK-8 (100 pM)-induced amylase release with a time course similar to that observed with VIP. Caffeine (20 mM) inhibited both amylase release and Ca2+ oscillations in response to CCK-8, suggesting that inhibition of Ca2+ oscillations does not necessarily lead to a potentiation of amylase release. When intracellular Ca2+ concentration ([Ca2+]c) was raised by thapsigargin (10 microM), a selective inhibitor of Ca(2+)-ATPase in the endoplasmic reticulum (ER), VIP (10 nM) induced significantly greater amylase release than that induced by VIP alone. When [Ca2+]c was lowered by preincubation with BAPTA-AM (25 microM), a cell-permeant Ca2+ chelator, VIP-induced amylase release was completely abolished. These results suggest that VIP, in spite of its inhibitory action on Ca2+ oscillations, facilitates a Ca(2+)-dependent process distal to the increase in [Ca2+]c to potentiate CCK-8-induced amylase release.

1. Unique spatiotemporal dynamics in cytosolic Ca2+ concentration, [Ca2+]c, were characterized in various cell types. In pancreatic acinar cells, physiological concentrations of cholecystokinin octapeptide, CCK-8, (10 pM) induce repetitive [Ca2+]c spikes commonly termed Ca2+ oscillation, whereas relatively higher concentrations (30 pM-1 nM) evoke biphasic [Ca2+]c dynamics; a rapid transient peak followed by a sustained increase. Much higher concentrations (1 nM) induce a large transient followed by a steep decay. 2. These [Ca2+]c dynamics correspond to secretory responses. Repetitive [Ca2+]c change is attributable to the upstroke of the bell-shaped dose-response relationship and the biphasic change is responsible for the downstroke of the relation (so called high-dose inhibited secretion). The large transient [Ca2+]c increase is associated with morphological changes such as bleb formation. 3. Possible interrelation between dose of secretagogues, secretory responses, [Ca2+]c dynamics, IP3 production, receptor occupation and morphological change will be discussed from both pharmacological and physiological points of view.

Isolated acini were stimulated with thapsigargin (TG), which is known to act selectively on Ca 2+ -ATPase in the endoplasmic reticulum (ER) and to inhibit Ca 2+ uptake into ER. Continuous stimulation with TG (0.02-2 μM) caused dose-dependent gradual rise in cytosolic Ca 2+ concentration, [Ca 2+ ] c , and in amylase release. The time course of TG-induced amylase release apparently resembled that of [Ca 2+ ] c . We found that there was a quantitative relation between the rate of rise in amylase release (Y) and that in [Ca 2+ ] c (X)

Rat pancreatic acini were isolated by collagenase digestion and exposed to cholecystokinin octapeptide (CCK-8) at concentrations of 0, 10 -10 and 10 -8 M. Secretion was monitored by measuring the release of amylase activity into the incubation medium that consisted of oxygenated HEPES-buffered Ringer's solution containing 2.5 mM Ca 2+ . The release of lactate dehydrogenase (LDH) into the incubation medium was measured to detect cell injury. The morphology of acinar cells was studied by light microscopy and scanning and transmission electron microscopy. A marked secretory response to stimulation with an optimal concentration of CCK-8 (10 -10 M) resulted in unaltered acinar cell morphology and in insignificantly increased LDH release as compared to non-stimulated controls

The effect of a receptor-mediated and voltage-dependent Ca 2+ entry blocker, SK&F 96365, on secretagogue-induced and spontaneous Ca 2+ dynamics was examined in isolated Fura-2loaded clusters of rat adrenal chromaffin cells. SK&F 96365 at 50 μM completely abolished 56 mM K + + or 10 μM nicotine-evoked [Ca 2+ ] c elevation. Stimulation with a muscarinic receptor agonist, methacholine (200 μM), induced [Ca 2+ ], dynamics consisted of the initial transient followed by a sustained plateau phase

The fluorescence of fura 2, a Ca2+-sensitive dye, is quenched in the presence of other divalent cations such as Mn2+. This characteristic of the dye provides a useful measure for

The effect of a receptor-mediated Ca2+ entry blocker, 1-(beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SKF 96365), on a secretagogue-induced secretory response and Ca2+ dynamics was examined in isolated acini of rat pancreas. SKF 96365 inhibited the secretory response induced by 100 pM cholecystokinin octapeptide (CCK-8) or 3 microM ethanaminium, 2-[(aminocarbonyl)oxy]-N,N,N-trimethyl,-chloride (carbachol) in isolated incubated acini up to 73.2 +/- 6.1% or 70.3 +/- 3.8% with IC50 of 47.7 microM or 42.0 microM, respectively. The inhibitory effect of SKF 96365 on the time courses of CCK-8-induced amylase release and [Ca2+]c (cytoplasmic Ca2+ concentration) elevation was further examined in isolated perifused acini or isolated acini loaded with Fura-2. In the 100 pM CCK-8-stimulated preparation, 30 microM SKF 96365 partly, and 100 microM SKF 96365 completely, inhibited the sustained plateau phase, but did not inhibit the initial phase of the Ca2+ and secretory responses. In the 5 pM CCK-8-stimulated preparation; (a) 30 microM SKF 96365 reduced the frequency of the [Ca2+]c oscillations, but caused little, if any, changes in the secretory response; (b) 100 microM SKF 96365 transformed the oscillatory [Ca2+]c dynamics to a transient increase followed by a gradual decay and caused a significant inhibition of the sustained secretory response. These results indicate that the sustained responses to secretagogues are dependent on a receptor-mediated Ca2+ entry which can selectively be blocked by SKF 96365 in rat pancreatic acini.

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 secretagogue were studied by electron microscopy 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(-6) M CCh stimulation, whereas the deformities of glandular tissues perfused via vessels were small even after 10(-3) M 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+.

1. Procaine (0.03-10 mM) inhibited carbachol (CCh)-induced amylase release from rat isolated pancreatic acini in a competitive manner. Kinetic analysis of the relation between CCh concentrations and the amount of amylase released in the presence of various procaine concentrations indicated that procaine caused competitive inhibition with the affinity constant (pA2) value of 5.00 +/- 0.08. 2. Receptor binding assay confirmed that procaine (0.01-10 mM) competitively inhibited [N-methyl-3H]-scopolamine chloride ([3H]-NMS) binding to its receptor with binding affinity (pKi) of 4.63 +/- 0.10. 3. Procaine transformed CCh-evoked [Ca2+]i dynamics: the initial rise in [Ca2+]i followed by a gradual decay during continuous stimulation with 3 microM CCh was transformed by 0.3 mM procaine to the oscillatory [Ca2+]i dynamics, which resembled the response to 0.3 microM CCh in the absence of procaine. The initial phase of [Ca2+]i oscillation corresponded to the initial phase of CCh-induced amylase release in isolated perfused acini. 4. Procaine (0.3-3 mM) did not inhibit the secretory response to cholecystokinin octapeptide (CCK-8) in isolated incubated acini. A higher concentration of procaine (10 mM) caused weak but significant inhibition of the response to only limited concentrations of CCK-8, 30 and 100 pM. Procaine lower than 10 mM was ineffective on [125I]-BH-CCK-8 binding, although procaine (10 mM) caused weak but significant inhibition of the binding.

1. The effects of chlorobutanol, a widely used drug preservative, on exocrine response and intracellular Ca2+ dynamics were examined in isolated pancreatic acini of the rat. 2. Chlorobutanol (1 mg ml-1) markedly inhibited the secretory response to cholecystokinin octapeptide (CCK-8), carbamylcholine chloride (carbachol), or sodium fluoride, a direct G-protein activator. However, chlorobutanol itself induced a maximal release of amylase when the dose was increased to 4 mg ml-1. 3. An oscillatory fluctuation of cytoplasmic Ca2+ concentration, [Ca2+]c, induced by 5 pM CCK-8 or 0.3 microM carbachol was totally abolished in the presence of 1 mg ml-1 chlorobutanol. 4. A biphasic change in [Ca2+]c induced by 100 pM CCK-8, a rapid rise followed by a gradual decay, was transformed to an oscillatory fluctuation by the preservative. 5. Chlorobutanol inhibited 13 pM [125I]-CCK-8 or 0.5 nM [3H]-methylscopolamine chloride binding to the acinar cells in a dose-dependent manner. 6. These results indicate that chlorobutanol produces discernible pharmacological effects on the secretory response in rat pancreatic acinar cells through changes in the Ca2+ dynamics. Possible sites of action could be at a binding process of secretagogues to their receptors, at an activation process of a G-protein located in the plasma membrane, or at the processes following G-protein activation. However, the possibility that the preservative may distort the Ca(2+)-transport function of the plasma membrane or the membrane of intracellular organella, especially Ca(2+)-sequestering pools, cannot be excluded.

Rat pancreatic acinar cells generate cytoplasmic Ca 2+ tides in a oscillatory manner in response to secretagogues at physiological concentrations. However, a fundamental controversy remains in identifying the site of onset and the direction of Ca 2+ gradient. Using a focal application technique and digital imaging, we now provide evidence which may resolve the controversy; the dose of carbachol as well as the topographical arrangement of an acinus may determine the site of onset and the direction of the Ca 2+ wave propagation

The order of inhibitory potency of local anesthetics in the carbachol-induced secretory response in rat pancreatic acini was tetracaine=procaine>dibucaine>ethyl-aminobenzoate>lidocaine. Almost the identical order of inhibitory potency was obtained in the binding of [N-methyl- 3 H]scopolamine to muscarinic receptors

Effects of chlorobutanol (ClBu), a widely used drug preservative, on intracellular Ca 2+ dynamics and catecholamine (CA) secretion were studied in perfused chromaffin cells of the rat adrenal gland. ClBu inhibited the CA secretion evoked by excess external potassium with IC 50 at 1.8 mM. This was ascribed to a blocking action of ClBu on Ca 2+ influx through voltage-dependent Ca 2+ channels

In the perfused cells isolated from the rat adrenal zona glomerulosa, deoxycorticosterone, aldosterone and dexamethasone (10 microM-1 mM) significantly inhibited elevation of the cytosolic concentration of calcium ion [Ca2+]c caused by a preceding stimulation with angiotensin II (ANG II) or high K+. The inhibition of [Ca2+]c was observed within a few minutes after application of the corticosteroids. The resting level of [Ca2+]c was reduced by aldosterone, whereas it was not significantly altered by deoxycorticosterone or dexamethasone. Since it is well established that the elevation of [Ca2+]c is required for the secretory responses to ANG II or high K+, the present results are compatible with a view that aldosterone secretion from the cortical cells in response to the secretagogues may, in turn, be attenuated by corticosteroids, including aldosterone itself.

We have provided novel evidence that CGA-LI is stored in the cells of granular convoluted tubule, and is secreted into saliva by stimulation with NAd in the isolated and perfused rat submandibular gland. The cells are also known to secrete various growth factors, which are released into blood stream. The cells may release CGA-LI into blood stream (endocrine secretion) in addition to salivary release (exocrine secretion). The endocrine- exocrine secretion of the cells resembles the secretion of renin in the cells of juxtaglomerular apparatus and coagulating gland.

In the study reported in this paper, sensitive ELISA for rat CgA was developed using synthetic rat CgA(359–389) as antigen, Nα-biotinylated glycylglycyl rat CgA(359–389), and antirat CgA(359–389) serum for the measurement of CgA-LI in rat saliva. CgA-LI in rat submandibular tissues and saliva was characterized by both immunohistochemical and immunochemical methods. Using isolated perfused rat submandibular gland. VIP at 0.1–1.0 nM in the presence of 0.1 μM ACh was found to cause CgA-LI secretion, whereas neither PACAP-27 nor PACAP-38 showed any effect on CgA secretion.

The effects of the oxidant tert-butylhydroperoxide (t-buOOH) on carbachol-stimulated pancreatic secretion in the vascularly perfused rat pancreas have been studied in parallel with [Ca2+]i signalling and amylase output in perifused rat pancreatic acinar cells. Perfusion of the pancreas with t-buOOH (0.1–1 mM) caused a rapid and irreversible inhibition of carbachol-stimulated (3×10−7 M) amylase and fluid secretion. Pre-perfusion of the pancreas with vitamin C and dithiothreitol or a cocktail of GSH and GSH-precursor amino acids provided only marginal protection against the deleterious effects of t-buOOH, even though GSH levels were elevated significantly. In perifused pancreatic acini, repetitive [Ca2+]i spikes evoked by carbachol (3×10−7 M) were sustained for 40 min. t-buOOH (1 mM) acutely increased the amplitude and duration of Ca2+ spikes, then attenuated Ca2+ spiking and subsequently caused a marked and sustained rise in [Ca2+]i. t-buOOH-induced alterations in carbachol-stimulated [Ca2+]i signalling and amylase release in perifused pancreatic acini were prevented by vitamin C. Although vitamin C restored impaired Ca2+ signalling and maintained amylase output in pancreatic acini, it seems likely that oxidative stress inhibits fluid secretion irreversibly in the intact pancreas, resulting in a loss of amylase output. Thus, perturbations in [Ca2+]i signalling may not fully explain the secretory block caused by oxidative stress in acute pancreatitis.

To clarify when the cholinergic receptor-mediated secretion mechanism of developing adrenal chromaffin cells is expressed and becomes functional, morphological changes and intracellular calcium dynamics were studied by immunohistochemistry, electron microscopy, and Fura-2 digital image analysis. From embryonic day 14 to 16, adrenal medullary cells were immunoreactive to noradrenaline-synthesizing enzyme (dopamine beta-hydroxylase) but not to adrenaline-synthesizing enzyme (phenylethanolamine N-methyltransferase). These cells contained either no granules or just a few granules of high electron density. Exocytotic figures were rarely observed in cells of the control or in cells after carbamylcholine stimulation. Nerve fibers in the adrenal medulla contained either no clear vesicles or very few. Neither methacholine nor nicotine caused a change of intracellular Ca2+ in most chromaffin cells. From embryonic day 18 to 20, chromaffin cells were immunoreactive to both dopamine beta-hydroxylase and phenylethanolamine N-methyltransferase and they contained relatively numerous secretory granules. Exocytotic figures were often seen in cells after carbamylcholine stimulation. The intra-adrenal nerve fibers contained numerous clear vesicles and a few dense-cored vesicles. Methacholine caused no rise of intracellular Ca2+, but nicotine induced a low to relatively high rise in many cells. From postnatal day 2 or 3 to postnatal week 1, numerous cells were immunoreactive to both dopamine beta-hydroxylase and phenylethanolamine N-methyltransferase, whereas some cells were reactive to dopamine beta-hydroxylase alone. Chromaffin cells were divisible into noradrenaline cells and adrenaline cells based on the ultrastructural features of their granules. Methacholine induced a moderate rise of intracellular Ca2+ and nicotine caused a high rise in many chromaffin cells, whereas, in some chromaffin cells, methacholine induced no rise of intracellular Ca2+ and nicotine induced a high rise. These results suggest that morphological changes of the developing cells and the intra-adrenal nerve fibers are related to the expression of a cholinergic receptor-mediated secretion mechanism and that this mechanism via a nicotinic receptor-mediated Ca2+ signaling pathway precedes the muscarinic receptor-mediated one during development.

The effects of a novel membrane-penetrable modulator, 2APB (2-aminoethoxy diphenyl borate), on Ins(1,4,5)P3-induced Ca2+ release were examined. 2APB inhibited Ins(1,4,5)P3-induced Ca2+ release from rat cerebellar microsomal preparations without affecting [3H]Ins(1,4,5)P3 binding to its receptor. The IC50 value (concentration producing 50% inhibition) of 2APB for inhibition of Ins(1,4,5)P3 (100 nM) induced Ca2+ release was 42 microM. Further increase in the concentration of 2APB (more than 90 microM) caused a gradual release of Ca2+ from cerebellar microsomal preparations. Addition of 2APB to the extracellular environment inhibited the cytosolic Ca2+ ([Ca2+]c) rise in intact cells such as human platelets and neutrophils stimulated by thromboxane-mimetic STA2 or thrombin, and leukotriene B4 (LTB4) or formyl-methionine-leucine-phenylalanine (FMLP), respectively. 2APB inhibited the contraction of thoracic aorta isolated from rabbits induced by angiotensin II (AII), STA2, and norepinephrine in a non-competitive manner, but showed no effect on the contraction of potassium-depolarized muscle. 2APB had no effect on the Ca2+ release from the ryanodine-sensitive Ca2+ store prepared from rat leg skeletal muscle and heart. Although the specificity of 2APB with respect to the intracellular signaling system was not fully established, 2APB is the first candidate for a membrane-penetrable modulator of Ins(1,4,5)P3 receptor, and it should be a useful tool to investigate the physiological role of the Ins(1,4,5)P3 receptor in various cells.

Background/Aims The intestinal epithelium is composed of various cells, and the enteric nervous system, which controls the epithelial functions, has different neurotransmitters and/or modulators. The aim of this study was to show whether the responses of intestinal epithelial cells to different neurotransmitters are elicited throughout the entire epithelium or are restricted to a certain cell. Methods The spatiotemporal dynamics of cytosolic calcium ion ([Ca 2+ ] c ) were measured by digital imaging analysis in isolated crypts of mouse ileum loaded with [1-[2-(5′-carboxyoxazol-2′-yl)-6-aminobenzofuran-5-oxy]-2-(2′-amino- 5′-ethylphenoxy)-ethane- N,N,N′,N′ -tetraacetic acid] pentakis (acetoxylmethyl) ester. Thereafter, the crypt cells were identified morphologically. Results Carbamylcholine elicited [Ca 2+ ] c dynamics in Paneth cells, showing a biphasic increase, but neither cholecystokinin octapeptide nor nicotine had any effect on the [Ca 2+ ] c of the crypt cells including the Paneth cells. Adrenaline and noradrenaline, but not isoproterenol, induced a transient elevation of [Ca 2+ ] c of some enterochromaffin cells. Increases in the [Ca 2+ ] c of most crypt cells were elicited by thapsigargin. Propagation of a [Ca 2+ ] c wave in the crypts was not evident. Conclusions Increases in [Ca 2+ ] c can be induced by carbamylcholine in Paneth cells and catecholamines in some enterochromaffin cells. The digital imaging analysis showed the heterogeneity of the responses of intestinal crypt cells to different transmitters.

Purpose: The cardinal role of Ca 2+ signaling in the development of heat damage at 42°C and 44°C and the effect of verapamil and diltiazem on the change of cytosolic Ca 2+ concentration, [Ca 2+ ] c were investigated. Methods and Materials: Hyperthermic treatment was performed by immersing the tubes containing the mouse mammary carcinoma FM3A cells in a water-bath set at 42.0°C or 44.0°C. The [Ca 2+ ] c of a single cell after hyperthermia was monitored by a digital image analyzing technique using Fura-2. Influx of Ca 2+ was examined by the measurement of the radioactivity of 45 Ca 2+ incorporated into the cells for 60 min during or after hyperthermia. Results: While the [Ca 2+ ] c of the cells treated at 37°C for 60 min was less than 230 nM, the percentage of the cells showing more than 230 nM increased after 42°C hyperthermia and that of the cells showing more than 300 nM increased after 44°C hyperthermia. When cellular uptake of 45 Ca 2+ during and after hyperthermia was examined, the increase in 45 Ca 2+ uptake was observed only for 44°C hyperthermia. Verapamil or diltiazem (100 μM) enhanced a delay of cell growth, an increase in [Ca 2+ ] c and an increase in 45 Ca 2+ influx for 42°C and 44°C hyperthermia. Conclusion: These results are compatible with the view that the increase in [Ca 2+ ] c after 42°C hyperthermia results from intracellular release from calcium store sites whereas the rise in [Ca 2+ ] c after 44°C hyperthermia is mainly due to entry of extracellular Ca 2+ . Verapamil or diltiazem combined with hyperthermia increased [Ca 2+ ] c , which may play a cardinal role in thermosensitization by these agents.