Tomio Kanno

Pituitary Adenylate Cyclase‐Activating Polypeptide Causes Ca2+ Release from Ryanodine/Caffeine Stores Through a Novel Pathway Independent of Both Inositol Trisphosphates and Cyclic AMP in Bovine Adrenal Medullary Cells

Abstract: Pituitary adenylate cyclase‐activating polypeptide (PACAP) causes both Ca2+ release and Ca2+ influx in bovine adrenal chromaffin cells. To elucidate the mechanisms of PACAP‐induced Ca2+ release, we investigated expression of PACAP receptors and measured inositol trisphosphates (IP3), cyclic AMP, and the intracellular Ca2+ concentration in bovine adrenal medullary cells maintained in primary culture. RT‐PCR analysis revealed that bovine adrenal medullary cells express the PACAP receptor hop, which is known to couple with both IP3 and cyclic AMP pathways. The two naturally occurring forms of PACAP, PACAP38 and PACAP27, both increased cyclic AMP and IP3, and PACAP38 was more potent than PACAP27 in both effects. Despite the effects of PACAP on IP3 production, the Ca2+ release induced by PACAP38 or by PACAP27 was unaffected by cinnarizine, a blocker of IP3 channels. The potencies of the peptides to cause Ca2+ release in the presence of cinnarizine were similar. The Ca2+ release induced by PACAP38 or by PACAP27 was strongly inhibited by ryanodine and caffeine. In the presence of ryanodine and caffeine, PACAP38 was more potent than PACAP27. PACAP‐induced Ca2+ release was unaffected by Rp‐adenosine 3′,5′‐cyclic monophosphothioate, an inhibitor of protein kinase A. Ca2+ release induced by bradykinin and angiotensin II was also inhibited by ryanodine and caffeine, but unaffected by cinnarizine. Although IP3 production stimulated by PACAP38 or bradykinin was abolished by the phospholipase C inhibitor, U‐73122, Ca2+ release in response to the peptides was unaffected by U‐73122. These results suggest that PACAP induces Ca2+ release from ryanodine/caffeine stores through a novel intracellular mechanism independent of both IP3 and cyclic AMP and that the mechanism may be the common pathway through which peptides release Ca2+ in adrenal chromaffin cells.

Salivary Secretion of Highly Concentrated Chromogranin a in Response to Noradrenaline and Acetylcholine in Isolated and Perfused Rat Submandibular Glands

Chromogranin A (CgA) is a member of a family of highly acidic proteins, chromogranins, which are co‐stored in the adrenergic neurons and paraneurons and co‐released with adrenaline and noradrenaline (NAd) in response to adequate stimulation. The present study provides novel evidence that CgA‐like immunoreactivity (IR) is stored in the exocrine cells in the granular convoluted tubule, and is secreted into saliva by stimulation with NAd and acetylcholine (ACh) in the isolated and perfused rat submandibular gland. NAd at 1 μM produced maximum secretion of CgA‐like IR (<< 0.9 mM) and a marked increase in salivary flow. Further increases in NAd concentration (10 or 100 μM) yielded concentration‐dependent decreases in both responses. ACh at 1 μM produced maximum salivary flow and a slight elevation of CgA‐like IR secretion (6 μM); 100 μM ACh decreased the salivary flow but increased the CgA‐like IR secretion (0.6 mM). Electron microscopic examination showed vigorous compound exocytosis of secretory granules in the cells of the granular convoluted tubule when the submandibular gland was stimulated with 1 μM NAd. These results provide an experimental basis for the view that the salivary CgA‐like IR secretion may be a sensitive and quantitative index of the activity of the sympathetic nervous system innervating the gland.

Photodynamic triggering of calcium oscillation in the isolated rat pancreatic acini

1 Photodynamic agents, due to their photon‐dependent selective activation, can selectively activate a number of physiological processes and may directly modulate signal transduction in a number of cells including pancreatic acinar cells. 2 Activation of the photodynamic agent sulphonated aluminium phthalocyanine (SALPC) triggered recurrent cytosolic calcium ([Ca2+]i) spiking in pancreatic acinar cells. 3 The photodynamically triggered calcium spiking could be blocked by phosphatidylinositol‐specific phospholipase C (PI‐PLC) inhibitor U73122, but not by phosphatidylcholine‐specific phospholipase C inhibitor D609. 4 Removal of extracellular Ca2+ abolished spiking, as did 2‐aminoethoxydiphenylborate (2‐APB), an inhibitory modulator of IP3‐mediated Ca2+ release from intracellular stores. 5 These data suggest that SALPC photodynamic action may permanently fix PI‐PLC in an active conformation, and this produced recurrent [Ca2+]i spiking.

Thyrotropin-releasing hormone injected intracerebroventricularly in the rat stimulates exocrine pancreatic secretion via the vagus nerve.

The effects of intracerebroventricular (i.c.v.) injection of synthetic thyrotropin-releasing hormone (TRH) and its analogue (gamma-butyrolactone-gamma-carbonyl-His-Pro-NH2) were tested in anesthetized rats fitted with pancreatic cannula. TRH injection induced dose-related increases in flow of pancreatic juice, protein output, and amylase output, each reaching a maximum within 10 min. Higher doses of TRH induced longer responses. Injection of the TRH analogue also caused dose-related secretory responses of the exocrine pancreas. The dose-related secretory responses to TRH and the TRH analogue were similar except that the responses to the highest dose of TRH analogue (1600 pmol/100 g b.w.) were significantly higher. Intravenous injection of TRH and the TRH analogue induced little, if any, secretory response of the exocrine pancreas. The effects of i.c.v. injection of TRH and the TRH analogue were completely abolished after bilateral subdiaphragmatic vagotomy. In addition to the secretory effects on the exocrine pancreas, i.c.v. injection of TRH and the analogue caused hyperglycemia, tachycardia, and tear secretion, but the intravenous injection of these peptides had no effect.

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.

ACUTE OXIDATIVE STRESS MODULATES SECRETION AND REPETITIVE CA2+ SPIKING IN RAT EXOCRINE PANCREAS

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 (3x10-7 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 (3x10-7 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.

A novel aspect of photodynamic action : induction of recurrent spikes in cytosolic calcium concentration

Effects of photodynamic action of gadolinium porphyrin‐like macrocycle B (PLMGdB) on cytosolic Ca2+ concentration, [Ca2+]c, was investigated in isolated rat pancreatic acini. The PLMGdB alone or light alone (2 min) had no effect on [Ca2+]c. Cell‐bound PLMGdB upon brief (0.5–2.0 min) light activation triggered recurrent spikes in [Ca2+]c. At lower PLMGdB concentration (100 nM) the spikes continued during the whole period of monitoring [Ca2+]c. At a higher concentration of 500 nM, the spikes continued for the first 40 min, followed by a gradual increase in basal [Ca2+]c upon which smaller spikes were superimposed. At 1, the spikes continued for the first 20 min, after that spiking gradually degenerated into a plateau phase. In many aspects, photodynamically triggered spikes resembled spikes generated by physiological concentrations of cholecystokinin. The spikes triggered by photodynamic action were likely to be the result of the ignition of a physiological “chain reaction”, because functional inositol‐1,4,5‐trisphosphate (IP3) receptors were required for spiking to occur. Two‐aminoe‐thoxydiphenylborate, an inhibitory modulator of IP3‐triggered Ca2+ release from intracellular stores, effectively inhibited photodynamically generated spikes. Therefore photodynamic action appears to be able to permanently transfix a physiological process, leading to long‐lasting pharmacological or therapeutic effects.

Stimulus-secretion coupling and Ca2+ dynamics in pancreatic acinar cells.

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.

Prevention of Acute Experimental Pancreatitis in Rats and Dogs by Intraduodenal Infusion of a Synthetic Trypsin Inhibitor

Acute hemorrhagic pancreatitis was induced in rats and dogs by a closed duodenal loop method. All dogs and most of the rats died of acute pancreatitis within 18 h after the operation. Infusion of a sy

Immunohistochemical Localization of Chromogranin A in the Acinar Cells of Equine Salivary Glands Contrasts with Rodent Glands

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.