The important discrepancy between the apparent affinity observed in Ca2+ mobilization studies and the Kd measured in binding studies is a consequence of the quantal process by which inositol 1,4,5-trisphosphate releases Ca2+ from bovine adrenal cortex microsomes

Canine cerebellar membranes were fractionated by differential centrifugation into a crude mitochondrial pellet (P2) and a crude microsomal pellet (P3). The effect of Mg2+ on inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release and [3H]IP3 binding was assessed. Mg2+ inhibited IP3-induced Ca2+ release in a concentration-dependent manner. Mg2+ influenced both the extent of IP3-induced Ca2+ release and the apparent affinity for IP3. A 10-fold change of free Mg2+ (from approximately 30 to approximately 300 microM) reduced the extent of Ca2+ release by two- to threefold and shifted the apparent Michaelis constant from approximately 0.5 to approximately 0.9 microM IP3. Thus Mg2+ seemed to be noncompetitive inhibitor of IP3-induced Ca2+ release. Mg2+ also inhibited Ca2+ release elicited by glycerophosphoinositol 4,5-bisphosphate, a poorly metabolized analogue of IP3. Mg2+ and heparin sodium were shown to be additive inhibitors of IP3-induced Ca2+ release. Mg2+ inhibited [3H]IP3 binding under experimental conditions designed to minimize IP3 hydrolysis. Scatchard plots indicated that 0.5 mM free Mg2+ reduced maximum binding from 10.9 to 3.5 pmol IP3 bound/mg protein and increased the dissociation constant from 136 to 227 nM. The modulation of [3H]IP3 binding and IP3-induced Ca2+ release by Mg2+ could be physiologically relevant.

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Effect of 1,25(OH)2D3 and its analogues on membrane phosphoinositide turnover and [Ca2+]i in Caco-2 cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1993.265.1.g143 ◽

1993 ◽

Vol 265 (1) ◽

pp. G143-G148 ◽

Cited By ~ 1

Author(s):

X. Y. Tien ◽

T. A. Brasitus ◽

B. M. Qasawa ◽

A. W. Norman ◽

M. D. Sitrin

Keyword(s):

Calcium Concentration ◽

Binding Affinities ◽

Similar Concentration ◽

Binding Studies ◽

Intact Cells ◽

Dihydroxyvitamin D3 ◽

Lower Affinity ◽

Rapid Action ◽

Inositol 1,4,5 Trisphosphate

Our laboratory has recently reported that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] rapidly stimulates membrane polyphosphoinositide turnover and increases intracellular calcium concentration ([Ca2+]i) in Caco-2 cells. The role of binding to the vitamin D receptor (VDR) in the regulation of these rapid biochemical events, however, remains unclear. The present studies were, therefore, conducted using analogues of 1,25(OH)2D3, which differ markedly in their affinities for the VDR, to assess and compare their effects on [Ca2+]i and on inositol 1,4,5-trisphosphate (IP3) formation. Competitive binding studies performed with both intact cells and high-salt cytosolic extracts from Caco-2 cells demonstrated that 1,25(OH)2D3 and 1,24-(OH)2-22-ene-24-cyclopropyl-D3 (BT) have high affinities for the VDR; 25(OH)-16-ene,23-yne-D3 (AT), however, has a much lower affinity (approximately 1,000-fold less) for the VDR. Despite these large differences in binding affinities for the VDR, AT and BT produced similar concentration-dependent increases in [Ca2+]i and in IP3 formation while 1,25(OH)2D3 was approximately 10-fold less active. These results indicate that the structural requirements for the rapid action of these secosteroids on signal transduction in Caco-2 cells are different from those for receptor binding and transcriptional regulation.

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Prostaglandin E2 inhibits secretagogue-induced enzyme secretion from rat pancreatic acini

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1991.260.5.g711 ◽

1991 ◽

Vol 260 (5) ◽

pp. G711-G719

Author(s):

J. Mossner ◽

R. Secknus ◽

G. M. Spiekermann ◽

C. Sommer ◽

M. Biernat ◽

...

Keyword(s):

Prostaglandin E2 ◽

Pancreatic Enzyme ◽

Enzyme Secretion ◽

Amylase Secretion ◽

Binding Studies ◽

Pancreatic Acini ◽

Inositol 1,4,5 Trisphosphate ◽

Pancreatic Enzyme Secretion ◽

Hcl Secretion ◽

Specific Receptors

Prostaglandins of the E type may have a potential role in pancreatic physiology and pathophysiology. Because prostaglandins of the E type inhibit HCl secretion in parietal cells via a specific receptor by inhibition of adenylylcyclase, we studied whether a similar mechanism exists in the exocrine pancreas. Isolated rat pancreatic acini were incubated with various concentrations of secretagogues, such as cholecystokinin-octapeptide (CCK-8), bombesin, carbachol, and vasoactive intestinal peptide (VIP), in the absence or presence of prostaglandin E2 (PGE2), and amylase secretion was measured. For receptor binding studies, acini and pancreatic membranes were incubated with [3H]PGE2 and either unlabeled PGE2 or other types of prostaglandins. PGE2 (10(-13) to 10(-5) M) did not inhibit basal amylase secretion. However, CCK-8-stimulated secretion was significantly inhibited. Stimulation of secretion by bombesin, carbachol, VIP, and secretin was also inhibited by PGE2, but not as pronounced as CCK-8-stimulated secretion. The formation of inositol 1,4,5-trisphosphate induced by CCK-8 was markedly inhibited by simultaneous incubation with PGE2. Furthermore, PGE2 slightly but significantly reduced the CCK-8-induced efflux of 45Ca2+ from prelabeled acini. Intact acini and a membrane fraction bound [3H]PGE2 and this function could be equally competed by either unlabeled PGE2 or PGE1 in contrast to less-related prostaglandins such as PGF2 alpha, PGD2, and prostacyclin. We conclude that prostaglandins of the E type inhibit pancreatic enzyme secretion stimulated by various secretagogues. This function is mediated via specific receptors for PGE. With regard to CCK-8-stimulated secretion this function may be mediated by an inhibition of formation of inositol 1,4,5-trisphosphate.

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Ryanodine and inositol trisphosphate receptors are differentially distributed and expressed in rat parotid gland

Biochemical Journal ◽

10.1042/bj3400519 ◽

1999 ◽

Vol 340 (2) ◽

pp. 519-527 ◽

Cited By ~ 27

Author(s):

Xuejun ZHANG ◽

Jiayu WEN ◽

Keshore R. BIDASEE ◽

Henry R. BESCH ◽

Richard J. H. WOJCIKIEWICZ ◽

...

Keyword(s):

Parotid Gland ◽

Binding Capacity ◽

Ruthenium Red ◽

Receptor Subtype ◽

Fluorescence Signal ◽

Binding Studies ◽

Inositol 1,4,5 Trisphosphate ◽

Basal Pole ◽

Rat Parotid ◽

Dual Activation

The present study examines the cellular distribution of the ryanodine receptor/channel (RyR) and inositol 1,4,5-trisphosphate receptor (InsP3R) subtypes in parotid acini. Using fluorescently labelled 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3-propionic acid glycyl-ryanodine (BODIPY™-ryanodine) and confocal microscopy, RyRs were localized primarily to the perinuclear region (basal pole) of the acinar cell. Ryanodine, Ruthenium Red, cAMP and cADP ribose (cADPR) competed with BODIPY-ryanodine, resulting in a reduction in the fluorescence signal. However, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] did not alter the binding of BODIPY-ryanodine. Using receptor-subtype-specific antisera, InsP3Rs (types I, II and III) were located predominantly in the apical pole of the parotid cell. The presence of these three subtypes was confirmed using reverse transcriptase PCR with RNA-specific oligonucleotide probes. Binding studies using a parotid cell-membrane fraction identified and characterized RyRs and InsP3Rs in terms of binding affinity (Kd) and maximum binding capacity (Bmax) and confirmed that cADPR displaces ryanodine from its binding sites. Ruthenium Red and 8-Br-cADP-ribose blocked Ca2+ release in permeabilized acinar cells in response to cADPR and cAMP or forskolin, whereas Ins(1,4,5)P3-induced Ca2+ release was unaffected. The localization of the RyRs and InsP3Rs in discrete regions endow broad areas of the parotid cell with ligand-activated Ca2+ channels. The consequences of the dual activation of the RyRs and InsP3Rs by physiologically relevant stimuli such as noradrenaline (norepinephrine) are considered in relation to Ca2+ signalling in the parotid gland.

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The effect of external calcium and pH on inositol trisphosphate-mediated calcium release from cerebellum microsomal fractions

Biochemical Journal ◽

10.1042/bj2580261 ◽

1989 ◽

Vol 258 (1) ◽

pp. 261-265 ◽

Cited By ~ 92

Author(s):

S K Joseph ◽

H L Rice ◽

J R Williamson

Keyword(s):

Calcium Release ◽

Feedback Inhibition ◽

Inhibitory Effect ◽

Scatchard Analysis ◽

Alkaline Ph ◽

Apparent Affinity ◽

Rat Cerebellum ◽

Low Concentrations ◽

Inositol 1,4,5 Trisphosphate ◽

External Calcium

Binding of D-myo-inositol 1,4,5-trisphosphate (InsP3) to rat cerebellum membranes has previously been shown to be stimulated by alkaline pH and inhibited by low concentrations of Ca2+ [Worley, Baraban, Suppatopone, Wilson & Snyder (1987) J. Biol. Chem. 262, 12132-12136]. In the present study, Scatchard analysis of InsP3 binding to cerebellum microsomes indicates that the effects of Ca2+ and pH are exerted through changes in the apparent affinity of the receptor without effects on maximal binding. The influence of extravesicular Ca2+ and pH on InsP3-mediated 45Ca2+ release was investigated. Extravesicular Ca2+ inhibited InsP3-mediated Ca2+ release. The inhibitory effect of Ca2+ was most marked when a sub-optimal concentration of InsP3 was used. An increase in extravesicular pH produced a decrease in the concentration of InsP3 that yielded half-maximal Ca2+ release. Regulation of the affinity of the InsP3 receptor by Ca2+ and pH can qualitatively account for the observed effects of these factors on InsP3-mediated Ca2+ release. Feedback inhibition of InsP3 binding by Ca2+ could provide a mechanism to generate Ca2+ oscillations, particularly under hormonal conditions that produce sub-optimal elevations of InsP3 concentration.

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Interaction of polyanions with the recognition sites for inositol 1,4,5-trisphosphate in the bovine adrenal cortex

European Journal of Pharmacology Molecular Pharmacology ◽

10.1016/0922-4106(91)90098-3 ◽

1991 ◽

Vol 208 (3) ◽

pp. 213-221 ◽

Cited By ~ 4

Author(s):

Thanh-Truc Luong ◽

Marc Poitras ◽

Guylain Boulay ◽

Gaétan Guillemette

Keyword(s):

Adrenal Cortex ◽

Recognition Sites ◽

Inositol 1,4,5 Trisphosphate

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Study on the stereoselectivity of Inositol 1,4,5-trisphosphate recognition sites of bovine adrenal cortex

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y92-055 ◽

1992 ◽

Vol 70 (4) ◽

pp. 434-441

Author(s):

Thanh-Truc Luong ◽

Guylain Boulay ◽

Gaétan Guillemette

Keyword(s):

Adrenal Cortex ◽

Receptor Interaction ◽

Microsomal Preparation ◽

Metabolizing Enzymes ◽

A Value ◽

Recognition Sites ◽

Naturally Occurring ◽

Inositol 1,4,5 Trisphosphate ◽

High Degree ◽

Hydrolysis Of

Inositol 1,4,5-trisphosphate (InsP3) is an intracellular messenger generated from the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C in response to Ca2+-mobilizing stimuli. InsP3 interacts with a specific receptor responsible for the release of sequestered Ca2+ from an intracellular store. The purpose of the present study was to evaluate the relative affinities of the naturally occurring D-isomer of InsP3 and that of its L-stereoisomer for the InsP3 receptor and the InsP3 metabolizing enzymes from bovine adrenal cortex. The InsP3 receptor recognized D- and L-isomers with respective affinities of 4.8 nM and 7.3 μM. This high degree of selectivity was also reflected in the capacity of both isomers to mobilize Ca2+ from the microsomal preparation. The partially purified InsP3 kinase preparation was also able to discriminate between the two stereoisomers. The activity of the kinase was half-maximally inhibited in the presence of 11 μM L-InsP3, a value much higher than the Km of the kinase for D-InsP3 (0.4 μM). Both stereoisomers exhibited equipotent affinities (around 17 μM) for the particulate preparation of InsP3 phosphatase. The enzyme, however, appeared to hydrolyze L-InsP3 at a much slower rate. These results demonstrated that the different recognition sites for InsP3 were expressing distinct levels of stereoselectivity. This property, which is an important aspect of ligand – receptor interaction, could be exploited for the design of new selective drugs interfering with InsP3 action and metabolism.Key words: inositol 1,4,5-trisphosphate (InsP3), InsP3 kinase, InsP3 phosphatase, InsP3 receptor, stereoselectivity, Ca2+ mobilization, adrenal cortex.

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Thiol-Reactive Agents Biphasically Regulate Inositol 1,4,5-Trisphosphate Binding and Ca2+ Release Activities in Bovine Adrenal Cortex Microsomes*

Endocrinology ◽

10.1210/endo.142.6.8195 ◽

2001 ◽

Vol 142 (6) ◽

pp. 2614-2621 ◽

Cited By ~ 9

Author(s):

Stéphane N. Poirier ◽

Marc Poitras ◽

Karina Laflamme ◽

Gaétan Guillemette

Keyword(s):

Adrenal Cortex ◽

Inositol 1,4,5 Trisphosphate ◽

Reactive Agents

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Muscarinic stimulation of gallbladder epithelium. I. Electrophysiology and signaling mechanisms

AJP Cell Physiology ◽

10.1152/ajpcell.1993.265.6.c1604 ◽

1993 ◽

Vol 265 (6) ◽

pp. C1604-C1612 ◽

Cited By ~ 5

Author(s):

G. A. Altenberg ◽

M. Subramanyam ◽

J. S. Bergmann ◽

K. M. Johnson ◽

L. Reuss

Keyword(s):

Phospholipase C ◽

Muscarinic Receptors ◽

Basolateral Membrane ◽

Receptor Activation ◽

Free Calcium ◽

Gallbladder Epithelium ◽

Binding Studies ◽

Necturus Gallbladder ◽

Inositol 1,4,5 Trisphosphate ◽

Cl Channels

To understand the effects of acetylcholine (ACh) on fluid-absorbing epithelia, we carried out experiments on Necturus gallbladder epithelium. Binding studies with 1-quinuclidinyl[phenyl-4(N)-3H]benzilate (QNB) demonstrated that Necturus gallbladder epithelial cells express high-affinity muscarinic receptors. The effects of ACh and carbachol were exerted from the basolateral surface and consisted of a transient hyperpolarization of both cell membranes and a concomitant decrease in the apparent fractional resistance of the apical membrane. Atropine blocked both effects. ACh also elicited transient elevations of inositol 1,4,5-trisphosphate and intracellular free calcium ([Ca2+]i) levels, the latter by both release from intracellular stores and basolateral influx. The phospholipase C antagonist U-73122 inhibited the effects of ACh, whereas inhibition of prostaglandin and guanosine 3',5'-cyclic monophosphate synthesis with indomethacin or methylene blue, respectively, had no effect. In conclusion, Necturus gallbladder epithelium expresses muscarinic receptors in the basolateral membrane. Receptor activation stimulates phospholipase C and elevates cellular levels of inositol 1,4,5-trisphosphate and [Ca2+]i. The elevation in [Ca2+]i activates K+ channels but apparently not Cl- channels.

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