scholarly journals Binding of inositol phosphates and induction of Ca2+ release from pituitary microsomal fractions

1987 ◽  
Vol 244 (2) ◽  
pp. 493-496 ◽  
Author(s):  
A Spät ◽  
G L Lukács ◽  
I Eberhardt ◽  
L Kiesel ◽  
B Runnebaum
Keyword(s):  
Anterior Pituitary ◽  
Inositol Phosphates ◽  
Maximal Effect ◽  
Reversible Binding ◽  
High Affinity ◽  
Inositol 1,4,5 Trisphosphate ◽  
Inositol Tetrakisphosphate

Bovine anterior-pituitary microsomal fractions exhibit high-affinity, saturable and reversible binding of inositol 1,4,5-[32P]trisphosphate; 50% of the labelled ligand is displaced by 3.5 nM-inositol 1,4,5-trisphosphate. 0.5 microM-inositol 1,4-bisphosphate and 10 microM-ATP. Inositol 1,4,5-trisphosphate induces the release of Ca2+ from the microsomal vesicles (half-maximal effect at 290 nM), and its action is potentiated by inositol tetrakisphosphate (half-maximal effect at 4 microM).

Effect of cholecystokinin on the accumulation of inositol phosphates in isolated pancreatic islets

1989 ◽  
Vol 257 (6) ◽  
pp. G865-G870
Author(s):  
J. Florholmen ◽  
D. Malm ◽  
B. Vonen ◽  
P. G. Burhol
Keyword(s):  
Insulin Secretion ◽  
Pancreatic Islets ◽  
Inositol Phosphates ◽  
Inositol Trisphosphate ◽  
Maximal Effect ◽  
Isolated Pancreatic Islets ◽  
Chromatography Analysis ◽  
Inositol Tetrakisphosphate ◽  
Per Se ◽  
Hydrolysis Of

Sulfated cholecystokinin octapeptide (CCK-8S) potentiated glucose-induced secretion in isolated pancreatic islets with a maximal effect at 12 mM glucose, whereas no effect was observed at 3.3 and 25 mM glucose. This effect of CCK-8S was maximal at 10(-7) M. Anion-exchange fast-protein liquid chromatography analysis of [3H]inositol phosphates derived from islets prelabeled with myo-[3H]inositol showed that glucose induced accumulation of the 1,4,5-isomer of inositol trisphosphate and of inositol tetrakisphosphate. At 3.3 mM glucose, CCK-8S stimulated accumulation of inositol trisphosphate and inositol tetrakisphosphate to levels induced by 25 mM glucose alone. The net effect of CCK-8S on the accumulation of the inositol phosphates was maximal at 12 mM glucose and decreased at higher glucose concentrations. At 12 mM glucose the accumulation of inositol phosphates increased gradually up to 10(-7) M CCK-8S. This study indicates that CCK-8S potentiates glucose-induced insulin secretion through a mechanism involving the hydrolysis of polyphosphoinositides and the generation of inositol phosphates. However, activation of the inositol cycle per se did not seem to induce insulin secretion.

scholarly journals Partial purification and some properties of rat brain inositol 1,4,5-trisphosphate 3-kinase

1988 ◽  
Vol 251 (1) ◽  
pp. 157-163 ◽  
Author(s):  
A J Morris ◽  
K J Murray ◽  
P J England ◽  
C P Downes ◽  
R H Michell
Keyword(s):  
Rat Brain ◽  
Inositol Phosphates ◽  
Gel Filtration ◽  
Partial Purification ◽  
First Order Kinetics ◽  
Inositol 1,4,5 Trisphosphate ◽  
Inositol Tetrakisphosphate ◽  
Dependent Protein ◽  
Metabolic Properties ◽  
Sepharose 4B

An enzyme which catalyses the ATP-dependent phosphorylation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was purified approx. 180-fold from rat brain cytosol by (NH4)2SO4 precipitation, chromatography through hydroxyapatite, anion-exchange fast protein liquid chromatography and gel-filtration chromatography. Gel filtration on Sepharose 4B CL gives an Mr of 200 × 10(3) for the native enzyme. The inositol tetrakisphosphate (InsP4) produced by the enzyme has the chromatographic, chemical and metabolic properties of Ins(1,3,4,5)P4. Ins(1,4,5)P3 3-kinase displays simple Michaelis-Menten kinetics for both its substrates, having Km values of 460 microM and 0.44 microM for ATP and Ins(1,4,5)P3 respectively. When many of the inositol phosphates known to occur in cells were tested, only Ins(1,4,5)P3 was a substrate for the enzyme; the 2,4,5-trisphosphate was not phosphorylated. Inositol 4,5-bisphosphate and glycerophosphoinositol 4,5-bisphosphate were phosphorylated much more slowly than Ins(1,4,5)P3. CTP, GTP and adenosine 5′-[gamma−thio]triphosphate were unable to substitute for ATP. When assayed under conditions of first-order kinetics, Ins(1,4,5)P3 kinase activity decreased by about 40% as the [Ca2+] was increased over the physiologically relevant range. This effect was insensitive to the presence of calmodulin and appeared to be the result of an increase in the Km of the enzyme for Ins(1,4,5)P3. Preincubation with ATP and the purified catalytic subunit of cyclic AMP-dependent protein kinase did not affect the rate of phosphorylation of Ins(1,4,5)P3 when the enzyme was assayed at saturating concentrations of Ins(1,4,5)P3 or at concentrations close to its Km for this substrate.

scholarly journals The high affinity state of inositol 1,4,5-trisphosphate receptor is a functional state.

1993 ◽  
Vol 268 (32) ◽  
pp. 24078-24082
Author(s):  
M Poitras ◽  
S Bernier ◽  
M Servant ◽  
D.E. Richard ◽  
G Boulay ◽  
...  
Keyword(s):  
Functional State ◽  
High Affinity ◽  
Inositol 1,4,5 Trisphosphate ◽  
Trisphosphate Receptor

scholarly journals Characterization of the inositol 1,4,5-trisphosphate-induced calcium release from permeabilized endocrine cells and its inhibition by decavanadate and p-hydroxymercuribenzoate

1989 ◽  
Vol 262 (1) ◽  
pp. 83-89 ◽  
Author(s):  
K J Föhr ◽  
J Scott ◽  
G Ahnert-Hilger ◽  
M Gratzl
Keyword(s):  
Endocrine Cells ◽  
Calcium Release ◽  
Cell Types ◽  
Inhibitory Effect ◽  
Maximal Effect ◽  
Thiol Compounds ◽  
Inositol 1,4,5 Trisphosphate ◽  
Dependent Inhibition ◽  
Pheochromocytoma Pc12 ◽  
First Time

The inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ compartment of endocrine cells was studied with alpha-toxin- and digitonin-permeabilized rat insulinoma (RINA2) and rat pheochromocytoma (PC12) cells. The Ca2+ uptake was ATP-dependent, and submicromolar concentrations of IP3 specifically released the stored Ca2+. Half-maximal Ca2+ release was observed with 0.25-0.5 mumol of IP3/l, and the amount of Ca2+ released due to IP3 could be enhanced by additional loading of the Ca2+ compartment. Consecutive additions of the same concentration of IP3 for 1-2 h always released the same amount of Ca2+ without desensitization, providing an ideal basis to further characterize the IP3-induced Ca2+ release. Here we describe for the first time a reversible inhibitory effect of decavanadate on the IP3-induced Ca2+ release. Among the vanadium species tested (decavanadate, oligovanadate and monovanadate), only decavanadate was inhibitory, with a half-maximal effect at 5 mumol/l in both cell types. The effect of decavanadate could be overcome by increasing the amount of sequestered Ca2+ or added IP3. Decavanadate did not affect the ATP-driven Ca2+ uptake but oligovanadate was inhibitory on Ca2+ uptake. p-Hydroxymercuribenzoate (pHMB) at concentrations between 10 and 30 mumol/l also inhibited the Ca2+ release due to IP3. Thiol compounds such as dithiothreitol (DTT; 1 mmol/l) added before pHMB removed all its inhibitory effect on the IP3-induced Ca2+ release, whereas the inhibition caused by decavanadate was unaffected by DTT. Thus, the decavanadate-dependent inhibition functions by a distinctly different mechanism than pHMB and could serve as a specific tool to analyse various aspects of the IP3-induced Ca2+ release within endocrine cells.

Substance P stimulation of polyphosphoinositide hydrolysis in rat anterior pituitary membranes involves a GTP-dependent mechanism

1991 ◽  
Vol 130 (1) ◽  
pp. 63-70 ◽  
Author(s):  
S. E. Mau ◽  
T. Saermark
Keyword(s):  
Substance P ◽  
Pertussis Toxin ◽  
Anterior Pituitary ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Sodium Deoxycholate ◽  
Pituitary Cells ◽  
Guanine Nucleotide ◽  
Nk1 Receptor ◽  
Gtp Binding

ABSTRACT Substance P (SP) stimulates polyphosphoinositide breakdown in the rat anterior pituitary through an NK-1 receptor. In the present study we present evidence that the coupling between the SP–NK1 receptor complex and polyphosphoinositide-specific phospholipase C (PI-PLC) in rat anterior pituitary membranes may involve a mechanism consistent with a GTP-binding protein. The formation of inositol phosphates from [3H]myo-inositol-labelled anterior pituitary membranes induced by SP was potentiated by GTP and non-hydrolysable guanine nucleotides. The stimulatory effects of SP alone and SP plus GTP could be blocked by addition of GDP-β-S (guanosine 5-O-(thiodiphosphate)) in excess. Basal and SP plus guanine nucleotide-induced inositol phosphate formation were stimulated by fluoride, whereas the effect of SP alone was inhibited. Pretreatment of anterior pituitary membranes with sodium deoxycholate attenuated the inositol phosphate response elicited by GTP and GTP-γ-S, whereas basal and SP-stimulated inositol phosphate production showed a peak at 1 mg sodium deoxycholate/ml. SP, fluoride and guanine nucleotide stimulatory effects on hydrolysis of polyphosphoinositide (PPI) were unaffected by pretreatment of anterior pituitary cells with cholera or pertussis toxin for 12 h. Treatment of anterior pituitary membranes with cholera and pertussis toxin yielded [32P]ADP-ribosylation of two proteins with molecular masses of 45 and 41 kDa respectively. We conclude that SP coupling to PI-PLC through the NK1 receptor in the rat anterior pituitary involves a GTP-binding mechanism distinct from the G-proteins associated with adenylate cyclase, Gs and Gi. Journal of Endocrinology (1991) 130, 63–70

GRP-preferring bombesin receptors increase generation of inositol phosphates and tension in rat myometrium

1993 ◽  
Vol 265 (6) ◽  
pp. C1579-C1587 ◽  
Author(s):  
F. Amiot ◽  
D. Leiber ◽  
S. Marc ◽  
S. Harbon
Keyword(s):  
Methyl Ester ◽  
Inositol Phosphates ◽  
Regulatory Proteins ◽  
Single Class ◽  
Gastrin Releasing Peptide ◽  
Uterine Contractions ◽  
High Affinity ◽  
Neuromedin B ◽  
Guanine Nucleotide Regulatory Proteins ◽  
Relative Potencies

In the estrogen-treated rat myometrium, bombesin (Bn) and related agonists triggered contraction and the increased generation of inositol phosphates. The relative order of potencies was identical for both responses: Bn = gastrin releasing peptide (GRP) = litorin = neuromedin C >> neuromedin B. Two specific GRP-preferring receptor antagonists, namely [D-Phe6]Bn-(6-13) methyl ester and [Leu14,psi 13-14]Bn were inhibitory for both Bn-mediated tension and generation of inositol phosphates. [125I-Tyr4]Bn bound to myometrial membranes with high affinity (Kd = 104 pM) to a single class of sites in a saturable and reversible manner. The relative potencies for inhibiting binding were GRP = litorin = [Tyr4]Bn (Ki = 0.4 to 0.6 nM) >> neuromedin B (Ki = 10.3 nM). The high affinity displayed by [D-Phe6]Bn-(6-13) methyl ester (Ki = 2.8 nM) and [Leu14,psi 13-14]Bn (Ki = 35 nM) for competing for [Tyr4]Bn binding supported the involvement of a GRP-preferring Bn receptor. Guanine nucleotides decreased the binding of [125I-Tyr4]Bn and accelerated the rate of ligand dissociation, reflecting the coupling of receptors to guanine nucleotide regulatory proteins (G proteins). The results demonstrate that rat myometrium expresses functional GRP-preferring Bn receptors whose activation stimulates the phospholipase C pathway, pertussis toxin-insensitive event that contributes to Bn-mediated uterine contractions.

Assessment of transport capacity of plasmalemmal Ca2+ pump in smooth muscle

1988 ◽  
Vol 255 (2) ◽  
pp. C226-C236 ◽  
Author(s):  
P. A. Lucchesi ◽  
R. A. Cooney ◽  
C. Mangsen-Baker ◽  
T. W. Honeyman ◽  
C. R. Scheid
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Membrane Vesicles ◽  
Reliable Estimate ◽  
Flux Rate ◽  
Transport Capacity ◽  
High Affinity ◽  
Transmembrane Flux ◽  
Inositol 1,4,5 Trisphosphate ◽  
Biochemical Studies

In resting smooth muscle, a variety of Ca2+ extrusion processes offset the inward Ca2+ leak. Biochemical studies suggest that the plasmalemmal Ca2+ pump dominates this process; however, this contention could not be proven without a reliable estimate of the inward Ca2+ leak that must be opposed by active transport. Recent studies using dispersed cells from the toad stomach provided such an estimate; thus we examined the capacity of the plasmalemmal Ca2+ pump in this tissue. Membranes were prepared using nitrogen cavitation, high-salt extraction, and flotation on discontinuous sucrose gradients. These membrane vesicles were enriched 16- to 24-fold for plasma membrane markers and exhibited an ATP-dependent uptake of 45Ca that was insensitive to azide or oxalate but sensitive to orthovanadate inhibition and calmodulin stimulation. 45Ca accumulated in the presence of ATP was rapidly released by Ca2+ ionophore but not by caffeine, inositol 1,4,5-trisphosphate, or GTP. Uptake exhibited a high affinity for Ca2+ (Km 0.2 microM) and a high-transport capacity, producing greater than 12,000-fold gradient for Ca2+ and a transmembrane flux rate greater than that observed in resting smooth muscle cells. Thus this enzyme is capable of maintaining steady-state Ca2+ levels in smooth muscle.

Role of external Na+ and cytosolic pH in agonist-evoked cytosolic Ca2+ response in human platelets

1994 ◽  
Vol 267 (6) ◽  
pp. C1543-C1552 ◽  
Author(s):  
M. Kimura ◽  
K. Nakamura ◽  
J. W. Fenton ◽  
T. T. Andersen ◽  
J. P. Reeves ◽  
...  
Keyword(s):  
Binding Sites ◽  
Human Platelets ◽  
Thrombin Receptor ◽  
Free Medium ◽  
Amidolytic Activity ◽  
Cytosolic Ph ◽  
Amino Acid Peptide ◽  
High Affinity ◽  
Inositol 1,4,5 Trisphosphate

The role of external Na+ in agonist-evoked platelet Ca2+ response is poorly understood. This was explored in this study. Removal of external Na+ decreased both cytosolic Ca2+ mobilization and external Ca2+ entry, induced by thrombin but not by ADP or vasopressin. That external Na+ regulates thrombin activities was demonstrated by 1) Na+ dependency of the amidolytic activity of thrombin, 2) inhibition of thrombin binding to the high-affinity binding sites in Na(+)-free medium, and 3) attenuation of thrombin-induced inositol 1,4,5-trisphosphate production in Na(+)-free medium. Moreover, Ca2+ response to the thrombin receptor 6-amino acid peptide was independent of external Na+. The role of external Na+ in modifying agonist-evoked Ca2+ response through activation of Na+/H+ antiport and cytosolic alkalinization was then explored. Cytosolic alkalinization by monensin or NH4Cl enhanced thrombin, ADP, and thimerosal-induced external Ca2+ entry. Thimerosal-induced acceleration of external Ca2+ entry was diminished by the inhibition of Na+/H+ antiport. Thus external Na+ enhances thrombin activities, and cytosolic pH mediates store-regulated external Ca2+ entry. However, Na+/H+ antiport activation is not essential for agonist-evoked Ca2+ mobilization and external Ca2+ entry.

scholarly journals Formation of methylphosphoryl inositol phosphates by extractions that employ methanol

1988 ◽  
Vol 253 (3) ◽  
pp. 703-710 ◽  
Author(s):  
J E Brown ◽  
M Rudnick ◽  
A J Letcher ◽  
R F Irvine
Keyword(s):  
Alkaline Phosphatase ◽  
Methanol Extract ◽  
Inositol Phosphates ◽  
Red Cell ◽  
Unknown Compound ◽  
Methyl Group ◽  
Phosphatidylinositol Bisphosphate ◽  
Inositol 1,4,5 Trisphosphate ◽  
Inositol Ring ◽  
Retinal Volume

Fixatives that contain methanol extract an unknown compound from several tissues including the retinas of squid (Loligo). We have determined that the compound probably contains (1) a myo-inositol ring that is phosphorylated in more than one position (including at the 5-hydroxyl), (2) a charged moiety that is not susceptible to alkaline phosphatase, and (3) a methyl group. We have found that the compound can be made by treating either phosphatidylinositol bisphosphate or human red cell ghosts with acidic methanol. We have confirmed the observation of Lips, Bross & Majerus [Proc. Natl. Acad. Sci. U.S.A. 85, 88-92] that the compound also can be made by methanolysis of inositol (cyclic 1:2,4,5)trisphosphate; however, we have not found inositol (cyclic 1:2,4,5)trisphosphate in either stimulated or unstimulated squid retinas. We tentatively identify the compound as (1-methylphosphoryl)inositol 4,5-bisphosphate formed by methanolysis of phosphatidylinositol 4,5-bisphosphate. By using this methanolysis to incorporate label from [14C]methanol, we have estimated the mass of inositol 1,4,5-trisphosphate in squid retinas to be approx. 30 mumol/l of retinal volume.

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