Physiological Secretagogues Research Articles

Effects of pine nodule extract and its component, SJ-2, on acetylcholine-induced catecholamine secretion and synthesis in bovine adrenal medullary cells

Extract of pine nodules (matsufushi) formed by bark proliferation on the surface of trees of Pinus tabulaeformis or Pinus massoniana has been used as an analgesic for joint pain, rheumatism, neuralgia, dysmenorrhea and other complaints in Chinese traditional medicine. Previous studies have reported that pine oil extract has analgesic and antitumor effects and a dissolving effect on gallstones (Anticancer research, 7:1153-1159, 1987). Extract of the oil from Siberian pine (Pinus sibirica) has been found to have an anti-inflammatory effect (J Nat Med., 62:436-440, 2008). The pharmacological effects of matsufushi extract, however, have not been studied in the sympathetic nervous system. Adrenal medullary cells are derived from embryonic neural crests and share many properties with sympathetic postganglionic neurons. In the present study, we report the effects of matsufushi extract and its components on catecholamine secretion and synthesis in cultured bovine adrenal medullary cells. We found that matsufushi extract (0.0003-0.005 %) and its component, SJ-2 (5-hydroxy-3-methoxy-trans-stilbene) (0.3-100 µM) concentration-dependently inhibited catecholamine secretion induced by acetylcholine, a physiological secretagogue. Matsufushi extract (0.0003-0.005 %) and SJ-2 (0.3-100 µM) also inhibited 45Ca2+ influx induced by acetylcholine in a concentration-dependent manner, similar to its effect on catecholamine secretion. They also suppressed 14C-catecholamine synthesis and tyrosine hydroxylase activity induced by acetylcholine. In Xenopus oocytes expressing α3β4 nicotinic acetylcholine receptors, matsufushi extract (0.00003-0.001 %) and SJ-2 (1-100 µM) directly inhibited the current evoked by acetylcholine. The present findings suggest that SJ-2, as well as matsufushi extract, inhibits acetylcholine-induced catecholamine secretion and synthesis by suppression of nicotinic acetylcholine receptor-ion channels in bovine adrenal medullary cells.

Effects of Sho-ju-sen, a Japanese herbal medicine, on catechoamine secretion induced by acetylcholine in cultured bovine adrenal medullary cells

Sho-ju-sen is a Japanese herbal medicine which has been used as a nourishing tonic. It is composed of a water extract of Kumazasa leaves (Sasa kurinensis Makino et Sibata) and ethanol extracts of Japanese red pine needles (Pinus densiflora Sieb. Et Zucc) and ginseng roots (Panax ginseng C. A. Meyer). Previous studies showed that Sho-ju-sen has antidepressant and anxiolytic effects in mice (Phytother Res., 15: 142-147, 2001 and 18: 173-176, 2004). Here we report that the effects of Sho-ju-sen on catecholamine secretion in cultured bovine adrenal medullary cells. Adrenal medullary cells have provided a good material for the detailed analysis of a drug's actions on catecholamine secretion and have been used as a model of the sympathetic neurons. We found that Sho-ju-sen (0.5 - 1 %) and extract of Kumazasa leaves (0.5-1 %), but not ethanol extract of Japanese red pine needles or ginseng roots, concentration-dependently inhibited the secretion of catecholamines induced by acetylcholine, a physiological secretagogue and agonist of nicotinic acetylcholine receptors. In Xenopus oocytes expressing α3β4 nicotinic acetylcholine receptors, Sho-ju-sen (0.5 - 1 %) directly inhibited Na+ current evoked by acetylcholine. Sho-ju-sen (0.3 - 1 %) also inhibited catecholamine secretion induced by veratridine, an activator of voltage-dependent Na+ channels, but not by 56 mM K+ which depolarizes cell membranes. The present findings demonstrate that Sho-ju-sen inhibits acetylcholine- and veratridine-induced catecholamine secretion by suppression of nicotinic acetylcholine receptor-ion channels and voltage-dependent Na+ channels in adrenal medulla cells and probably sympathetic neurons, and also suggest that Sho-ju-sen has an anti-stress effect.

Inhibitory effects of pine nodule extract and its component, SJ-2, on acetylcholine-induced catecholamine secretion and synthesis in bovine adrenal medullary cells

Extract of pine nodules (matsufushi) formed by bark proliferation on the surface of trees of Pinus tabulaeformis or Pinus massoniana has been used as an analgesic for joint pain, rheumatism, neuralgia, dysmenorrhea and other complaints in Chinese traditional medicine. Here we report the effects of matsufushi extract and its components on catecholamine secretion and synthesis in cultured bovine adrenal medullary cells. We found that matsufushi extract (0.0003–0.005%) and its component, SJ-2 (5-hydroxy-3-methoxy-trans-stilbene) (0.3–100 μM), but not the other three, concentration-dependently inhibited catecholamine secretion induced by acetylcholine, a physiological secretagogue. Matsufushi extract (0.0003–0.005%) and SJ-2 (0.3–100 μM) also inhibited 45Ca2+ influx induced by acetylcholine in a concentration-dependent manner, similar to its effect on catecholamine secretion. They also suppressed 14C-catecholamine synthesis and tyrosine hydroxylase activity induced by acetylcholine. In Xenopus oocytes expressing α3β4 nicotinic acetylcholine receptors, matsufushi extract (0.00003–0.001%) and SJ-2 (1–100 μM) directly inhibited the current evoked by acetylcholine. The present findings suggest that SJ-2, as well as matsufushi extract, inhibits acetylcholine-induced catecholamine secretion and synthesis by suppression of nicotinic acetylcholine receptor-ion channels in bovine adrenal medullary cells.

Ikarisoside A inhibits acetylcholine-induced catecholamine secretion and synthesis by suppressing nicotinic acetylcholine receptor-ion channels in cultured bovine adrenal medullary cells.

Ikarisoside A is a natural flavonol glycoside derived from plants of the genus Epimedium, which have been used in Traditional Chinese Medicine as tonics, antirheumatics, and aphrodisiacs. Here, we report the effects of ikarisoside A and three other flavonol glycosides on catecholamine secretion and synthesis in cultured bovine adrenal medullary cells. We found that ikarisoside A (1-100μM), but not icariin, epimedin C, or epimedoside A, concentration-dependently inhibited the secretion of catecholamines induced by acetylcholine, a physiological secretagogue and agonist of nicotinic acetylcholine receptors. Ikarisoside A had little effect on catecholamine secretion induced by veratridine and 56mMK(+). Ikarisoside A (1-100μM) also inhibited (22)Na(+) influx and (45)Ca(2+) influx induced by acetylcholine in a concentration-dependent manner similar to that of catecholamine secretion. In Xenopus oocytes expressing α3β4 nicotinic acetylcholine receptors, ikarisoside A (0.1-100μM) directly inhibited the current evoked by acetylcholine. It also suppressed (14)C-catecholamine synthesis and tyrosine hydroxylase activity induced by acetylcholine at 1-100μM and 10-100μM, respectively. The present findings suggest that ikarisoside A inhibits acetylcholine-induced catecholamine secretion and synthesis by suppression of nicotinic acetylcholine receptor-ion channels in bovine adrenal medullary cells.

New Insights Into the Pharmacological Potential of Plant Flavonoids in the Catecholamine System

Flavonoids are biologically active polyphenolic compounds widely distributed in plants. Recent research has focused on high dietary intake of flavonoids because of their potential to reduce the risks of diseases such as cardiovascular diseases, diabetes, and cancers. We report here the effects of plant flavonoids on catecholamine signaling in cultured bovine adrenal medullary cells used as a model of central and peripheral sympathetic neurons. Daidzein (0.01 – 1.0 μM), a soy isoflavone, stimulated 14C-catecholamine synthesis through plasma membrane estrogen receptors. Nobiletin (1.0 – 100 μM), a citrus polymethoxy flavone, enhanced 14C-catecholamine synthesis through the phosphorylation of Ser19 and Ser40 of tyrosine hydroxylase, which was associated with 45Ca2+ influx and catecholamine secretion. Treatment with genistein (0.01 – 10 μM), another isoflavone, but not daidzein, enhanced [3H]noradrenaline uptake by SK-N-SH cells, a human noradrenergic neuroblastoma cell line. Daidzein as well as nobiletin (≥ 1.0 μM) inhibited catecholamine synthesis and secretion induced by acetylcholine, a physiological secretagogue. The present review shows that plant flavonoids have various pharmacological potentials on the catecholamine system in adrenal medullary cells, and probably also in sympathetic neurons.

A fundamental role for NO-PLC signaling pathway in mediating intracellular Ca 2+ oscillation in pancreatic acini

The aim of the present study was to investigate the possible interaction between intracellular Ca 2+ and nitric oxide (NO) in rat pancreatic acinar cells, especially intracellular signaling events. (1) Nitric oxide donors SNP (0.1–100 μM) and NOR-3 (50–400 μM) induced Ca 2+ oscillations in fluo-4-loaded acini, that appeared to be analogous to what we usually observe in acini stimulated with physiological secretagogues such as CCK-8 and this oscillations were abolished in the presence of carboxy-PTIO. (2) The NO donors-evoked Ca 2+ oscillations were not abolished even in the absence of extracellular Ca 2+ but totally disappeared when cells were pretreated with thapsigargin, a sarcoplasmic-endoplasmic reticulum Ca 2+ ATPase (SERCA) inhibitor. (3) Inhibition of guanylate cyclase with 1 H-[1,2,4] oxadiazolo [4,3-a] quinoxaline-1-one (ODQ) attenuated Ca 2+ oscillations evoked by SNP in the absence of extracellular Ca 2+. (4) Inhibitors of phospholipase C activity, U73122 and the IP 3R blocker xestospongin C, both abolished the SNP-induced Ca 2+ response. (5) Furthermore, we found that both CCK-8 and carbachol (CCh) induced NO production in DAF-2-loaded acinar cells and that an inhibitor of NO synthase, N G-monomethyl- l-arginine (L-NMMA), significantly reduced CCK-8-induced Ca 2+ oscillation. These results indicate that NO mobilizes Ca 2+ from internal stores through activation of guanylate cyclase and resultant cGMP production. In addition, PLC activation of IP 3 production is also suggested to be involved in Ca 2+ mobilization via IP 3 receptors. This suggests the presence of cross-talk between Ca 2+ and NO in pancreatic acini and this cascade may, at least partially, participate in physiological secretagogue-evoked Ca 2+ dynamics in pancreatic acinar cells.

ALDOSTERONE-PRODUCING ADENOMA CAUSING HUMAN PRIMARY ALDOSTERONISM ARE CHARACTERISED BY UNDER- EXPRESSION OF THE TWIK-RELATED ACID-SENSITIVE K+ CHANNEL 2 (TASK-2) GENE: PP.21.321

Background: Primary aldosteronism is a common cause of arterial hypertension, but its underlying molecular mechanisms are unknown. However, it is well known that K+ is a key regulator of aldosterone secretion, which interacts with physiological secretagogues of aldosterone as angiotensin II (Ang II) and endothelin-1. Genetic manipulation of a widely distributed channels class, which generate background or “leak” potassium (K+) currents, the Twik-related Acid-Sensitive K channel 1 (TASK-1) and 3 (TASK-3), generated a phenotype that mimics human primary aldosteronism. Objective: To measure the expression of the TASK family in a series of aldosterone producing adenoma (APA). Methods: Whole transcriptome of 24 APA was compared to that of a normal adrenocortical tissue pool (n = 10) by oligomicroarray technique. Data were validated by quantitative real time RT PCR. Immunohistochemistry was used to investigate TASKs protein expression by adrenocortical tissues. Results and Conclusions: The most expressed TASK channel gene in both the normal ZG and in APAs was TASK-1, followed by TASK-2 while the expression of TASK-3 was scant. With a whole transcriptome analysis followed by confirmation with quantitative real time RT-PCR we found a consistent and marked under expression of TASK-2 channel in all studied APA (n = 24), while TASK-1 and TASK-3 were heterogeneously expressed, as compared to the normal adrenal cortex. Immunohistochemistry was used to confirm the expression of the 3 TASK K+ channels in the adrenal cortex at the protein level and quantitative immnunoblotting will be used to confirm the differences detected at the transcript level. The underexpression of TASK-2 channels in APA suggests blunted leak” K+ currents, with ensuing lowering of the membrane potential and thereby increased likelihood of opening of the voltage-gated T-type calcium channels. The latter is known to render the adrenocortical zona glomerulosa cells more sensitive to aldosterone secretagogues. Hence, our ex vivo findings, while pointing to TASK-2 channels under-expression as an important mechanism of primary aldosteronism, mandates further studies to ascertain the molecular mechanisms by which this underexpression develops.

Mast Cells and Arachidonic Acid Cascade in Inflammation

Prostaglandin D2 PGD2 is a major cyclooxygenase metabolite of arachidonic acid produced by mast cells and it is released following allergen challenge in diseases, such as allergic diseases. PGD2 may act as a neuromodulator and as an allergic and inflammatory mediator. In allergic diseases, activated mast cell synthesizes prostaglandin D2 (first cyclo-oxygenate mediator) which has bronchoconstrictive and vasodilating effects and attracts several leukocytes. It has been found that activated mast cells, challenged with physiological and non- physiological secretagogues, release elevated histamine and tryptase and chymase, leukotrienes B4, C4 and D4, 5-hydroxyeicosatetraenoic acid, PGD2, Platelet Activating Factor (PAF), heparin, and high-molecular-weight neutrophil chemotactic factor and cytokines/chemokines. PGD2 exerts its biological activity through the DP and CRTH2 receptors and their cDNA cloning which were characterized 15 years ago. In this report, we revisited the biological effects of arachidonic acid compounds released by activated mast cells in allergic and inflammatory states.

Vasoactive Intestinal Peptide Increases Cystic Fibrosis Transmembrane Conductance Regulator Levels in the Apical Membrane of Calu-3 Cells through a Protein Kinase C-Dependent Mechanism

Noncholinergic neurons contribute to innate airway defenses by releasing vasoactive intestinal peptides (VIP), which stimulates the submucosal glands to produce a bicarbonate-rich fluid containing mucins and antimicrobial factors. VIP elevates cAMP and activates cystic fibrosis transmembrane conductance regulator (CFTR) channels; however, its effects on surface expression have not been investigated. We studied CFTR levels in the apical membrane of polarized Calu-3 cell monolayers, a widely used model for submucosal gland serous cells. Biotinylation during VIP exposure revealed a significant increase in apical CFTR within 10 min, which reached a maximal 3.3-fold increase after 30 min. Total CFTR content of cell lysates was not altered during this time period; therefore, the increase in surface CFTR reflects redistribution from intracellular pools. Internalization assays revealed that apical accumulation was due, at least in part, to a reduction in the rate of CFTR endocytosis. VIP-induced accumulation of apical CFTR was mimicked by phorbol ester but not by forskolin, and it was blocked by the protein kinase (PK)C inhibitors bisindolylmaleimide X (BisX) or chelerythrine chloride but not by the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89). Increases in surface expression were paralleled by enhanced iodide effluxes during cAMP stimulation. BisX inhibition of VIP responses was abrogated when monolayers were pretreated with tannic acid to inhibit endosome recycling. Thus, PKC increases the surface expression of CFTR channels in addition to potentiating their responsiveness to PKA phosphorylation. Integrated regulation through multiple signaling pathways may be a common feature of VIP and other physiological secretagogues.

Twenty-Four Hour Continuous Ghrelin Infusion Augments Physiologically Pulsatile, Nycthemeral, and Entropic (Feedback-Regulated) Modes of Growth Hormone Secretion

Ghrelin is a 28-amino acid acylated peptide that potentiates GHRH stimulation and opposes somatostatin inhibition acutely. Whether prolonged ghrelin administration can sustain physiological patterns of GH secretion remains unknown. Continuous delivery of ghrelin will amplify physiological patterns of GH secretion over 24 h. Men and women ages 29-69 yr, body mass indices 23-52 kg/m2, were included in the study. The study was performed at an academic medical center. Twenty-four hour continuous sc infusion of saline vs. ghrelin (1 microg/kg.h) with frequent sampling was examined. Deconvolution and entropy analyses were performed. IGF-I concentrations were determined. Basal, pulsatile, nycthemeral, and entropic measures of GH secretion were calculated. Ghrelin infusion compared with saline infusion for 24 h elevated (median) acylated ghrelin, GH, and IGF-I concentrations by 8.1-fold (P < 0.001),11-fold (P < 0.001), and 1.4-fold (P = 0.002). GH secretory-burst mass and frequency increased by 6.6-fold (P = 0.004) and 1.7-fold (P < 0.001), respectively, resulting in a 12-fold increase in pulsatile GH secretion (P < 0.001). Interpulse variability decreased significantly (P = 0.046), whereas GH secretory-burst shape and half-life did not change. The amplitude of the nycthemeral GH rhythm increased by 3.4-fold (P < 0.001), and GH patterns became more irregular (higher approximate entropy P < 0.001). Combining GHRH with ghrelin was not an additive in driving GH secretion. Continuous ghrelin infusion for 24 h elevates acylated ghrelin, GH and IGF-I concentrations, and stimulates pulsatile, nycthemeral, and entropic modes of GH secretion. The consistency of outcomes in a heterogeneous cohort of adults suggests potentially broad utility of this physiological secretagogue in hyposomatotropic states.

Detection and quantification of ppb level potassium in biological samples in the presence of high sodium by ion chromatographic method

Employing a silica gel column modified with carboxyl groups and an eluent consisting of 4.0 mM tartaric acid and 0.75 mM dipicolinic acid in 2% acetone, an otherwise difficult quantification of K + at ppb level in presence of 6000 ppm NaCl was achieved by incorporating 0.75 mM 18-crown-6 ether in the mobile phase and subtracting the blank NaCl signal from each chromatogram. Optimized analytical conditions were established in terms of relative standard deviation (%) of retention time, peak area and calibration equations, and also by peak asymmetry factor. The net efflux of K + into the gastric lumen under in vitro conditions of acid secretion was investigated in Ussing chamber model. The effects of the physiological secretagogue histamine and the antisecretory agents cimetidine, omeprazole and SCH28080 were studied. The decline of K + efflux in presence of cimetidine, and the rise of the same in the presence of omeprazole and SCH28080 were conspicuously discernible, thereby validating the usefulness of ion chromatography based K + quantification method under biological experimental conditions.

TOXIC GLOBALISATION OF SECRETAGOGUE-ELICITED ACINAR CELL CA2+ SIGNALING AND PREVENTION BY ATP OR REMOVAL OF CA2+ FROM THE CELL EXTERIOR

Background & Aims: We have previously demonstrated that low concentrations of the pancreatic acinar cell toxins taurolithocholic-3-sulphate (TLCS) or palmitoleic acid ethyl ester (POAEE) induce toxic globalisation of inositol trisphosphate (IP3)-mediated signaling increases in the cytosolic calcium ion concentration ([Ca2+]C, IP3 applied via patch pipette). We sought to determine whether toxic globalisation of [Ca2+]C can occur with physiological secretagogues, and how it can be prevented. Methods: Isolated mouse pancreatic acinar cells were examined by confocal microscopy to measure changes in [Ca2+]C (Fluo4-AM), mitochondrial function (NADH autofluorescence), ATP concentration (Mg Green) and cell fate (propidium iodide, PI). Cholecystokinin (CCK, 1-20 pM) or acetylcholine (ACh, 50 nM) were perfused extracellularly and low concentrations (10 mM) of TLCS or POAEE added, while whole-cell recordings of Ca2+-dependent Cl− currents were made. In some experiments, supplementary ATP was added to the internal pipette solution. Results: CCK and ACh elicited typical [Ca2+]C signals in the granular pole with stimulus-metabolism coupling (NADH). Extracellular TLCS or POAEE (10 mM) transformed these signals into prolonged (>30 s), global [Ca2+]C increases accompanied by NADH and ATP depletion, inducing necrosis. Supplementary ATP reduced the period of globalisation associated with each elevation of [Ca2+]C and prevented necrosis, as did removal of Ca2+ from the external medium, and in the case of POAEE, hydrolase inhibition, preventing POA formation. Conclusion: Physiological Ca2+ signals elicited by secretagogues can undergo toxic globalisation from low concentrations of acinar cell toxins, resulting in mitochondrial Ca2+ overload, inhibition of ATP production and cell death. Cell injury could be prevented by blockade of Ca2+ entry.

Dual Effects of Daidzein, a Soy Isoflavone, on Catecholamine Synthesis and Secretion in Cultured Bovine Adrenal Medullary Cells

We recently demonstrated the occurrence and functional roles of plasma membrane estrogen receptors in cultured bovine adrenal medullary cells. Here we report the effects of daidzein, a phytoestrogen of soybeans, on catecholamine synthesis and secretion in the cells. Incubation of cells with daidzein for 20 min increased the synthesis of (14)C-catecholamines from [(14)C]tyrosine but not [(14)C]dihydroxyphenylalanine, in a concentration-dependent manner (10-1000 nm). The stimulatory effect of daidzein on (14)C-catecholamine synthesis was not inhibited by ICI182,780, a classical estrogen receptor inhibitor. Acetylcholine, a physiological secretagogue, stimulated the synthesis of (14)C-catecholamines, which was suppressed by daidzein at 1 mum. Daidzein at high concentrations (1-100 microm) suppressed catecholamine secretion induced by acetylcholine. Furthermore, daidzein (10-1000 nm) inhibited the specific binding of [(3)H]17beta-estradiol to plasma membranes isolated from bovine adrenal medulla. The present findings suggest that daidzein at low concentrations stimulates catecholamine synthesis through plasma membrane estrogen receptors but at high concentrations inhibits catecholamine synthesis and secretion induced by acetylcholine in bovine adrenal medulla. The latter effect of daidzein may be a beneficial action on the cardiovascular system.

Distinct K+ conductive pathways are required for Cl− and K+ secretion across distal colonic epithelium

Secretion of Cl(-) and K(+) in the colonic epithelium operates through a cellular mechanism requiring K(+) channels in the basolateral and apical membranes. Transepithelial current [short-circuit current (I(sc))] and conductance (G(t)) were measured for isolated distal colonic mucosa during secretory activation by epinephrine (Epi) or PGE(2) and synergistically by PGE(2) and carbachol (PGE(2) + CCh). TRAM-34 at 0.5 microM, an inhibitor of K(Ca)3.1 (IK, Kcnn4) K(+) channels (H. Wulff, M. J. Miller, W. Hänsel, S. Grissmer, M. D. Cahalan, and K. G. Chandy. Proc Natl Acad Sci USA 97: 8151-8156, 2000), did not alter secretory I(sc) or G(t) in guinea pig or rat colon. The presence of K(Ca)3.1 in the mucosa was confirmed by immunoblot and immunofluorescence detection. At 100 microM, TRAM-34 inhibited I(sc) and G(t) activated by Epi ( approximately 4%), PGE(2) ( approximately 30%) and PGE(2) + CCh ( approximately 60%). The IC(50) of 4.0 microM implicated involvement of K(+) channels other than K(Ca)3.1. The secretory responses augmented by the K(+) channel opener 1-EBIO were inhibited only at a high concentration of TRAM-34, suggesting further that K(Ca)3.1 was not involved. Sensitivity of the synergistic response (PGE(2) + CCh) to a high concentration TRAM-34 supported a requirement for multiple K(+) conductive pathways in secretion. Clofilium (100 microM), a quaternary ammonium, inhibited Cl(-) secretory I(sc) and G(t) activated by PGE(2) ( approximately 20%) but not K(+) secretion activated by Epi. Thus Cl(-) secretion activated by physiological secretagogues occurred without apparent activity of K(Ca)3.1 channels but was dependent on other types of K(+) channels sensitive to high concentrations of TRAM-34 and/or clofilium.

Improvement in Subject's Worst Symptom associated with Treatment of 2.0% Diquafosol Tetrasodium in two Studies of Dry Eye Disease

The aim of the present study was to investigate the possible interaction between intracellular Ca2+ and nitric oxide (NO) in rat pancreatic acinar cells, especially intracellular signaling events. (1) Nitric oxide donors SNP (0.1–100 μM) and NOR-3 (50–400 μM) induced Ca2+ oscillations in fluo-4-loaded acini, that appeared to be analogous to what we usually observe in acini stimulated with physiological secretagogues such as CCK-8 and this oscillations were abolished in the presence of carboxy-PTIO. (2) The NO donors-evoked Ca2+ oscillations were not abolished even in the absence of extracellular Ca2+ but totally disappeared when cells were pretreated with thapsigargin, a sarcoplasmic-endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor. (3) Inhibition of guanylate cyclase with 1 H-[1,2,4] oxadiazolo [4,3-a] quinoxaline-1-one (ODQ) attenuated Ca2+ oscillations evoked by SNP in the absence of extracellular Ca2+. (4) Inhibitors of phospholipase C activity, U73122 and the IP3R blocker xestospongin C, both abolished the SNP-induced Ca2+ response. (5) Furthermore, we found that both CCK-8 and carbachol (CCh) induced NO production in DAF-2-loaded acinar cells and that an inhibitor of NO synthase, NG-monomethyl-l-arginine (L-NMMA), significantly reduced CCK-8-induced Ca2+ oscillation. These results indicate that NO mobilizes Ca2+ from internal stores through activation of guanylate cyclase and resultant cGMP production. In addition, PLC activation of IP3 production is also suggested to be involved in Ca2+ mobilization via IP3 receptors. This suggests the presence of cross-talk between Ca2+ and NO in pancreatic acini and this cascade may, at least partially, participate in physiological secretagogue-evoked Ca2+ dynamics in pancreatic acinar cells.

Can Tears be replaced by Aqueous Humor in very Dry Eyes?

The aim of the present study was to investigate the possible interaction between intracellular Ca2+ and nitric oxide (NO) in rat pancreatic acinar cells, especially intracellular signaling events. (1) Nitric oxide donors SNP (0.1–100 μM) and NOR-3 (50–400 μM) induced Ca2+ oscillations in fluo-4-loaded acini, that appeared to be analogous to what we usually observe in acini stimulated with physiological secretagogues such as CCK-8 and this oscillations were abolished in the presence of carboxy-PTIO. (2) The NO donors-evoked Ca2+ oscillations were not abolished even in the absence of extracellular Ca2+ but totally disappeared when cells were pretreated with thapsigargin, a sarcoplasmic-endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor. (3) Inhibition of guanylate cyclase with 1 H-[1,2,4] oxadiazolo [4,3-a] quinoxaline-1-one (ODQ) attenuated Ca2+ oscillations evoked by SNP in the absence of extracellular Ca2+. (4) Inhibitors of phospholipase C activity, U73122 and the IP3R blocker xestospongin C, both abolished the SNP-induced Ca2+ response. (5) Furthermore, we found that both CCK-8 and carbachol (CCh) induced NO production in DAF-2-loaded acinar cells and that an inhibitor of NO synthase, NG-monomethyl-l-arginine (L-NMMA), significantly reduced CCK-8-induced Ca2+ oscillation. These results indicate that NO mobilizes Ca2+ from internal stores through activation of guanylate cyclase and resultant cGMP production. In addition, PLC activation of IP3 production is also suggested to be involved in Ca2+ mobilization via IP3 receptors. This suggests the presence of cross-talk between Ca2+ and NO in pancreatic acini and this cascade may, at least partially, participate in physiological secretagogue-evoked Ca2+ dynamics in pancreatic acinar cells.

A store-operated mechanism determines the activity of the electrically excitable glucagon-secreting pancreatic α-cell

The glucagon-releasing pancreatic α-cells are electrically excitable cells but the signal transduction leading to depolarization and secretion is not well understood. To clarify the mechanisms we studied [Ca 2+] i and membrane potential in individual mouse pancreatic α-cells using fluorescent indicators. The physiological secretagogue l-adrenaline increased [Ca 2+] i causing a peak, which was often followed by maintained oscillations or sustained elevation. The early effect was due to mobilization of Ca 2+ from the endoplasmic reticulum (ER) and the late one to activation of store-operated influx of the ion resulting in depolarization and Ca 2+ influx through voltage-dependent L-type channels. Consistent with such mechanisms, the effects of adrenaline on [Ca 2+] i and membrane potential were mimicked by inhibitors of the sarco(endo)plasmic reticulum Ca 2+ ATPase. The α-cells express ATP-regulated K + (K ATP) channels, whose activation by diazoxide leads to hyperpolarization. The resulting inhibition of the voltage-dependent [Ca 2+] i response to adrenaline was reversed when the K ATP channels were inhibited by tolbutamide. However, tolbutamide alone rarely affected [Ca 2+] i, indicating that the K ATP channels are normally closed in mouse α-cells. Glucose, which is the major physiological inhibitor of glucagon secretion, hyperpolarized the α-cells and inhibited the late [Ca 2+] i response to adrenaline. At concentrations as low as 3 mM, glucose had a pronounced stimulatory effect on Ca 2+ sequestration in the ER amplifying the early [Ca 2+] i response to adrenaline. We propose that adrenaline stimulation and glucose inhibition of the α-cell involve modulation of a store-operated current, which controls a depolarizing cascade leading to opening of L-type Ca 2+ channels. Such a control mechanism may be unique among excitable cells.

Trafficking of mutant carboxypeptidase E to secretory granules in a beta-cell line derived from Cpe(fat)/Cpe(fat) mice.

We have reinvestigated the stability and intracellular routing of mutant carboxypeptidase E in NIT3 cells, a pancreatic beta-cell line derived from the Cpe(fat)/Cpe(fat) mouse. Pulse-chase experiments demonstrated that this protein has a half-life of approximately 3 h in these cells and that up to 45% of the proCPE(202) can escape degradation by the proteosome. In double-label immunofluorescence microscopy, a portion of the mutant CPE did not colocalize with calnexin, an endoplasmic reticulum marker, but was found in prohormone convertase 2-containing secretory granules, demonstrating that it had escaped degradation and arrived at a post-Golgi compartment. The mutant CPE as well as prohormone convertase 2 were secreted into the medium in a stimulated manner by treatment with the physiological secretagogue, glucagon-like peptide-1, consistent with its presence in granules of the regulated secretory pathway. The presence of mutant carboxypeptidase E in granules supports a potential role for its involvement as a sorting/retention receptor in the trafficking of proinsulin to the regulated secretory pathway.

Insulin receptor signaling and sarco/endoplasmic reticulum calcium ATPase in beta-cells.

Glucose is the main physiological secretagogue for insulin secretion by pancreatic beta-cells, and the major biochemical mechanisms involved have been elucidated. In particular, an increase in intracellular calcium is important for insulin exocytosis. More recently, it has become apparent that the beta-cell also has many of the elements of the insulin receptor signal transduction pathway, including the insulin receptor and insulin receptor substrate (IRS) proteins 1 and 2. Studies with transgenic models have shown that the beta-cell-selective insulin receptor knockout and the IRS-1 knockout lead to reduced glucose-induced insulin secretion. Overexpression of the insulin receptor and IRS-1 in beta-cells results in increased insulin secretion and increased cytosolic Ca(2+). We have thus postulated the existence of a novel autocrine-positive feedback loop of insulin on its own secretion involving interaction with the insulin receptor signal transduction pathway and regulation of intracellular calcium homeostasis. Our current working hypothesis is that this glucose-dependent interaction occurs at the level of IRS-1 and the sarco(endo)plasmic reticulum calcium ATPase, the calcium pump of the endoplasmic reticulum.

Correlation between electrical activity and intracellular Ca2+ oscillations in GH3 rat anterior pituitary cells

Simultaneous measurements of electrical activity and intracellular Ca2+ levels were performed in perforated-patch current-clamped individual GH3 cells. Both in cells showing brief (<100 ms) and long action potentials (APs), we found a good correlation between the averaged intracellular Ca2+ concentration ([Ca2+]i) and AP frequency, but not between the mean [Ca2+]i and AP duration. Nevertheless, the magnitude of spontaneous Ca2+ oscillations was highly dependent on the size and duration of the APs. The decay of the Ca2+ transients was not slowed when the size of the oscillations was varied either spontaneously or after elongation of the AP with the K+ channel blocker tetraethyl ammonium. Furthermore, the recovery from Ca2+ loads similar to those induced by the APs was slightly retarded after treatment of the cells with intracellular store Ca2+-ATPase inhibitors. Among previous results showing that caffeine-induced [Ca2+]i increases are secondary to electrical activity enhancements in GH3 cells, these data indicate that the Ca2+ entry triggered via APs is the primary determinant of the [Ca2+]i variations, and that Ca2+-induced Ca2+ release has a minor contribution to Ca2+ oscillations recorded during spontaneous activity. They also point to modulation of electrical activity patterns as a crucial factor regulating spontaneous [Ca2+]i signalling, and hence pituitary cell functions in response to physiological secretagogues.