scholarly journals Hormone-induced protein phosphorylation. I. Relationship between secretagogue action and endogenous protein phosphorylation in intact cells from the exocrine pancreas and parotid.

1982 ◽  
Vol 95 (3) ◽  
pp. 903-908 ◽  
Author(s):  
S D Freedman ◽  
J D Jamieson
Keyword(s):  
Protein Phosphorylation ◽  
Exocrine Pancreas ◽  
Second Messengers ◽  
Intact Cells ◽  
Cholecystokinin Octapeptide ◽  
Phosphorylation Of Proteins ◽  
Endogenous Protein ◽  
Dose Dependent Fashion ◽  
Dose Dependent

We undertook studies to determine whether secretagogue action on the exocrine pancreas and parotid is accompanied by phosphorylation of proteins in intact cells. For this purpose, rat pancreatic, and parotid lobules were preincubated with 32Pi for 45 min at 37 degrees C, washed, and then incubated at 37 degrees C in the presence or absence of secretagogues that effect discharge through different second messengers. Among a variety of polypeptides exhibiting enhanced phosphorylation in pancreatic lobules upon a 30-s incubation in the presence of the secretagogues carbamylcholine, cholecystokinin octapeptide, or secretin, one species with an Mr of 29,000 was especially notable for three reasons: (a) its enhanced level of phosphorylation was dependent on the dose of secretagogue used and was still apparent after incubation for 30 min at 37 degrees C; (b) an analogous phosphorylated polypeptide was observed in isoproterenol-stimulated parotid lobules; and (c) in both tissues its selective dephosphorylation was observed upon termination of stimulation by administration of atropine to carbamylcholine-stimulated pancreatic lobules and propranolol to isoproterenol-stimulated parotid lobules. These results suggest that the phosphorylation of one protein with an Mr of 29,000 is closely correlated both temporally and in a dose-dependent fashion with secretagogue action in both the exocrine pancreas and parotid.

scholarly journals Hormone-induced protein phosphorylation. III. regulation of the phosphorylation of the secretagogue-responsive 29,000-dalton protein by both Ca2+ and cAMP in vitro.

1982 ◽  
Vol 95 (3) ◽  
pp. 918-923 ◽  
Author(s):  
S D Freedman ◽  
J D Jamieson
Keyword(s):  
Protein Kinase ◽  
Protein Phosphorylation ◽  
Exocrine Pancreas ◽  
Second Messengers ◽  
Subcellular Fractions ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Intact Cells ◽  
Dependent Protein

In the preceding papers, we demonstrated that the endogenous phosphorylation of a 29,000-dalton protein is stimulated in response to secretagogue application to intact cells from the rat exocrine pancreas and parotid and dephosphorylated upon termination of secretagogue action. One- and two-dimensional gel analysis of 32Pi-labeled pancreatic and parotid lobules as well as their respective subcellular fractions revealed that the same protein was covalently modified in both tissues and was localized to the ribosomal fraction. To identify the intracellular second messengers which may mediate or modulate the phosphorylation of the 29,000-dalton protein in intact cells, the effects of Ca2+, cAMP, and cGMP on the endogenous phosphorylation of this protein were assessed in subcellular fractions from the rat pancreas and parotid. Our results demonstrate that the phosphorylation of the 29,000-dalton polypeptide may be regulated by both Ca2+ and cAMP in the pancreas and in the parotid. No cGMP-dependent protein phosphorylation was found in either tissue. As in the in situ phosphorylation studies, the Ca2+- and cAMP-dependent phosphorylation of this same protein was localized to the ribosomal fraction. The cAMP-dependent protein kinase activity was found primarily in the postmicrosomal supernatant in contrast to the Ca2+-dependent protein kinase that appeared to be tightly associated with the substrate in addition to being present in the postmicrosomal supernatant. The data suggest that, in cells from the exocrine pancreas and parotid, secretagogues may regulate the phosphorylation of the 29,000-dalton protein through Ca2+ and/or cAMP.

scholarly journals Sodium-proton exchange stimulates Ca2+ release from acidocalcisomes of Trypanosoma brucei

1996 ◽  
Vol 315 (1) ◽  
pp. 265-270 ◽  
Author(s):  
Anibal E. VERCESI ◽  
Roberto DOCAMPO
Keyword(s):  
Trypanosoma Brucei ◽  
Proton Exchange ◽  
Dramatic Decrease ◽  
Intact Cells ◽  
Sodium Proton Exchange ◽  
Dose Dependent Fashion ◽  
Na Effect ◽  
Dose Dependent ◽  
Acidic Compartments ◽  
Ionophore Monensin

Acidocalcisomes are acidic vacuoles present in trypanosomatids that contain a considerable fraction of intracellular Ca2+ [Vercesi, Moreno and Docampo (1994) Biochem. J. 304, 227–233; Scott, Moreno and Docampo (1995) Biochem. J. 310, 789–794; Docampo, Scott, Vercesi and Moreno (1995) Biochem. J. 310, 1005–1012]. The data presented here indicate that Na+ stimulates Ca2+ release from the acidocalcisomes of digitonin-permeabilized Trypanosoma brucei procyclic trypomastigotes in a dose-dependent fashion, this effect being enhanced by increasing pH of the medium from 7.0 to 7.8. The hypothesis that this Na+ effect was mediated by alkalinization of the acidocalcisomes via a Na+/H+ antiporter was supported by experiments showing that Na+ promotes release of Acridine Orange previously accumulated in these vacuoles. This putative antiporter did not transport Li+ and was not sensitive to the amiloride analogue 5-(N-ethyl-N-isopropyl)amiloride. Addition of the Na+/H+ ionophore monensin to intact cells loaded with fura 2, in the nominal absence of extracellular Ca2+ to preclude Ca2+ entry, was followed by an increase in cytosolic Ca2+ concentration ([Ca2+]i), which was more accentuated in the presence of extracellular Na+. An increase in intracellular pH (pHi) of BCECF-loaded cells was detected after addition of monensin in the presence of extracellular Na+, whereas a dramatic decrease in pHi was detected in its absence, thus indicating the presence of a significant amount of releasable protons in the acidic compartments. These results are consistent with the presence of a Na+/H+ antiporter in the acidocalcisomes that could be involved in the regulation of pHi and [Ca2+]i in these parasites.

Regulation of digestion. II. Effects of insulin and glucagon on pancreatic secretion

1984 ◽  
Vol 246 (4) ◽  
pp. G451-G456
Author(s):  
H. C. Tseng ◽  
J. H. Grendell ◽  
S. S. Rothman
Keyword(s):  
Exocrine Pancreas ◽  
Digestive Enzyme ◽  
Celiac Artery ◽  
Portal Circulation ◽  
Amylase Output ◽  
Endocrine Hormones ◽  
Dose Dependent Fashion ◽  
High Concentrations ◽  
Dose Dependent ◽  
Islet Cell Hormones

The endocrine islet-cell hormones insulin and glucagon are secreted at high concentrations into an intrapancreatic portal circulation and have been reported to affect the secretion of digestive enzyme by the exocrine pancreas. In the present experiments, insulin and glucagon were injected into the celiac artery of anesthetized rats to evaluate their effects on the secretion of amylase and trypsinogen by the pancreas. Neither hormone when given alone significantly changed the output of either enzyme. However, when given with the pancreatic secretagogue cholecystokinin, each altered the effect of injection of cholecystokinin. In a dose-dependent fashion insulin increased trypsinogen output without affecting amylase output, whereas glucagon inhibited amylase output and left trypsinogen output unchanged. Thus, both hormones produced a more trypsinogen-dominant pancreatic juice than that observed with cholecystokinin alone, although in different ways. These findings suggest that the endocrine hormones insulin and glucagon may regulate secretion of digestive enzymes by the pancreas by modulating the response to stimuli of overall protein secretion such as cholecystokinin.

INCREASED SENSITIVITY OF A PLATELET-SUBPOPULATION TO CRYOPRESERVATION

1987 ◽  
Author(s):  
M van Marwijk-Kooy ◽  
A C Dullemond-Westland ◽  
H C van Prooijen ◽  
M I Riemens ◽  
J W N Akkerman
Keyword(s):  
Protein Phosphorylation ◽  
Cell Damage ◽  
Second Messengers ◽  
Membrane Damage ◽  
Human Platelets ◽  
Activation Mechanism ◽  
Small Cells ◽  
High Dose ◽  
Intact Cells ◽  
Secretion Granules

When cryopreserved human platelets are stimulated with a high dose of collagen (40 μg/ml), about 50% of the cells fail to aggregate, as measured by counting the disappearance of single platelets. This suggests the presence of a subpopulation with increased susceptibility to cryopreservation-induced cell damage. In an attempt to identify this subfraction fresh platelet suspensions were separated into four size-dependent fractions by counterflow centrifugation. Large platelets (MPV 11.1 μm3 ) better preserved their responsiveness than small platelets (4.2 μm3) with intermediate responses for fractions of 5.7 and 8.0 μm3. In contrast, the partial loss of secretion granules (βTG and PF-4) seen during cryopreservation was greater in the large platelets than in the small cells and did not parallel the differences in aggregability.In a second separation technique the unresponsive fraction was separated from the cells that aggregated with collagen. This fraction consisted of intact cells (LDH), and normal secretion granules (βTG-PF-4) but failed to show changes in Pl-cycle and protein phosphorylation, two essential steps in the activation mechanism of platelets. In accordance with this, the total cryopreserved suspension showed a partial reduction in these second messengers with a 35% lower phosphatidic acid formation, 50-55% less protein phosphorylation and a 50-75% reduction (depending on stimulus concentration) of the liberation of intracellular Ca2+ ions, compared with fresh platelets.Hence, cryopreservation disturbs the activation mechanism of a platelet subpopulation consisting of about 50% of the cells, probably as a result of membrane damage, whereas the other half of the population recovers relatively unharmed from the freeze-thawing procedure.Supported by the Praeventiefund (grant 28953), The Hague.

ACTION OF GTPγS [GUANOSINE 5∲-0-(3-THIOPHOSPHATE)] ON SAPONIN-PERMEABILISED PLATELETS: INVOLVEMENT OF 'G' PROTEINS IN PLATELET ACTIVATION

1987 ◽  
Author(s):  
K S Authi ◽  
B J Evenden ◽  
N Crawford
Keyword(s):  
G Protein ◽  
Shape Change ◽  
Second Messengers ◽  
Phosphatidyl Inositol ◽  
Protein S ◽  
Human Platelets ◽  
Dependent Manner ◽  
Guanine Nucleotide Binding Protein ◽  
Intact Cells ◽  
Dose Dependent

Certain ligand-receptor interactions at cell surfaces lead to the phospholipase-C (PLC) hydrolysis of phosphatidyl inositol (4.5) bisphosphate (PIP2). The products serve as intracellular second messengers, e.g. inositol (1.4.5) trisphosphate (IP3) releases Ca2+ from intracellular stores and diacylglycerol activates protein kinase-C. From studies using GTP and analogues (e.g. GTPγS) there is evidence of a key role for a guanine nucleotide binding protein(s) as a link between receptors and PIP2 hydrolysis. We report the actions of GTPγS on washed human platelets permeabilised with saponin (12-14 μg/ml) to allow penetration of low MWt polar substances. The responses to GTPγS are dose dependent (range 9-60 μM) and at 60 μM the agent induces shape change, aggregation and the secretion of 50% of previously incorporated [14C]-5HT. No effect of GTPγS is seen with intact cells. Shape change occurs 25-30 sec after GTPγS; aggregation and secretion is complete after 3 min. When GTP was used (up to 135 μM) with similarly permeabilised platelets no responses were initiated. Phosphatidylinositol turnover was monitored using 32P-labelling before permeabilisation. The addition of 90 μM GTPγS resulted in a 143 ± 23% (n=4) increase in 32P-phosphatidic acid (PA) with respect to the basal levels of “saponised control” cells. These findings suggest that GTPγS stimulates PLC activity through a ‘G’ protein interaction. The GDP analogue (GDPβS) produced no activation responses in saponised platelets but inhibited responses induced by GTPγS in a dose dependent manner (0-480 μM, max inhibition 480 μM). At 960 μM, GDPβS totally inhibited aggregation and secretion initiated by low doses of thrombin (0.1 U/ml) and collagen (1 μg/ml). Identical inhibition by GDPβS of thrombin and collagen-induced activation of intact platelets was observed indicating membrane penetration of this analogue. Shape change effects were not inhibited by GDPSS. The inhibitory effects of GDPSS towards thrombin and collagen induced secretion could be progressively overcome at higher doses of thrombin (0.2 U/ml - 2 U/ml) and collagen (5 μg/ml - 60 μg/ml) suggesting that at higher concentrations these agonists may exert effects through 'G' protein-independent mechanisms.

Cholecystokinin octapeptide decreases food intake in white-crowned sparrows

1993 ◽  
Vol 264 (5) ◽  
pp. R852-R856
Author(s):  
R. D. Richardson ◽  
T. Boswell ◽  
S. C. Weatherford ◽  
J. C. Wingfield ◽  
S. C. Woods
Keyword(s):  
Food Intake ◽  
Receptor Antagonist ◽  
Mammalian Species ◽  
Meal Size ◽  
Dark Cycle ◽  
Cholecystokinin Octapeptide ◽  
Dose Dependent Fashion ◽  
Cck Receptor Antagonist ◽  
Dose Dependent ◽  
Short Days

White-crowned sparrows maintained on short days (9:15-h light-dark cycle) were peripherally injected with 1.0, 4.0, and 16 micrograms/kg ip of cholecystokinin octapeptide (CCK-8). Meal size over the subsequent 30 min was significantly depressed in a dose-dependent fashion. Water intake was not affected. The anorexic effect caused by 4.0 micrograms/kg was attenuated by 100 micrograms/kg of the type-A CCK receptor antagonist MK-329 but not by 300 micrograms/kg of the type-B CCK receptor antagonist L 365,260, suggesting that CCK-induced suppression of food intake in this species is mediated by a CCK-A receptor. Administration of both CCK-A and CCK-B receptor antagonists alone resulted in no change in meal size. These experiments suggest that white-crowned sparrows, when weight stable, respond to CCK-8 in a manner comparable with several mammalian species.

scholarly journals Inositol trisphosphate formation and calcium mobilization in Swiss 3T3 cells in response to platelet-derived growth factor

1984 ◽  
Vol 222 (1) ◽  
pp. 195-201 ◽  
Author(s):  
M J Berridge ◽  
J P Heslop ◽  
R F Irvine ◽  
K D Brown
Keyword(s):  
Growth Factor ◽  
Second Messengers ◽  
Platelet Derived Growth Factor ◽  
Maximal Effect ◽  
3T3 Cells ◽  
Intact Cells ◽  
Swiss 3T3 ◽  
Phosphatidylinositol 4 ◽  
Dose Dependent ◽  
Hydrolysis Of

Swiss 3T3 cells incubated for 60 h with [3H]inositol incorporated radioactivity into phosphatidylinositol (PI) and the two polyphosphoinositides phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2). On stimulation with platelet-derived growth factor (PDGF) there were significant increases in the levels of inositol 1-phosphate (IP1), inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3). The effect of PDGF and IP3 on Ca2+ mobilization was studied in both intact cells and in ‘leaky’ cells that had been permeabilized with saponin. In intact cells, PDGF stimulated the efflux of 45Ca2+, whereas IP3 had no effect. Conversely, IP3 stimulated 45Ca2+ efflux from ‘leaky’ cells, which were insensitive to PDGF. ‘Leaky’ cells, which accumulated 45Ca2+ to a steady state within 20 min, were found to release approx. 40% of the label within 1 min after addition of 10 microM-IP3. This stimulation of 45Ca2+ release by IP3 was reversible and was also dose-dependent, with a half-maximal effect at approx. 0.3 microM. It seems likely that an important action of PDGF on Swiss 3T3 cells is to stimulate the hydrolysis of PIP2 to form IP3 and diacylglycerol, both of which may function as second messengers. Our results indicate that IP3 mobilizes intracellular Ca2+, and we propose that diacylglycerol may act through C-kinase to activate the Na+/H+ antiport. By generating two second messengers, PDGF can simultaneously elevate the intracellular level of Ca2+ and alkalinize the cytoplasm by lowering the level of H+.

Plasminogen Interactions with Immobilized Fibrinogen

1989 ◽  
Vol 62 (04) ◽  
pp. 1078-1082 ◽  
Author(s):  
Burt Adelman ◽  
Patricia Ouynn
Keyword(s):  
Immunosorbent Assay ◽  
Aminocaproic Acid ◽  
Enzyme Linked Immunosorbent Assay ◽  
Binding Regions ◽  
Dose Dependent Fashion ◽  
Epsilon Aminocaproic Acid ◽  
Dose Dependent

SummaryThis report describes the binding of plasminogen to fibrinogen adsorbed onto polystyrene wells. Binding was determined by enzyme linked immunosorbent assay. Both glu- and lys-plasminogen bound to immobilized fibrinogen in a dose-dependent fashion. However, more lys- than glu-plasminogen bound when equal concentrations of either were added to immobilized fibrinogen. Plasminogen binding was inhibited by epsilon aminocaproic acid indicating that binding was mediated via lysine-binding regions of plasminogen. Soluble fibrinogen added in excess of immobilized fibrinogen did not compete for plasminogen binding but fibrinogen fragments produced by plasmin digestion of fibrinogen did. Treatment of immobilized fibrinogen with thrombin caused a small but significant (p <0.01) increase in plasminogen binding. These studies demonstrate that immobilized fibrinogen binds both glu- and lys-plasminogen and that binding is mediated via lysine-binding regions. These interactions may facilitate plasminogen binding to fibrinogen adsorbed on to surfaces and to cells such as platelets which bind fibrinogen.

Soluble Thrombomodulin Purified from Human Urine Exhibits a Potent Anticoagulant Effect In Vitro and In Vivo

1995 ◽  
Vol 73 (05) ◽  
pp. 805-811 ◽  
Author(s):  
Yasuo Takahashi ◽  
Yoshitaka Hosaka ◽  
Hiromi Niina ◽  
Katsuaki Nagasawa ◽  
Masaaki Naotsuka ◽  
...  
Keyword(s):  
Human Urine ◽  
Specific Activity ◽  
Anticoagulant Activity ◽  
Rabbit Lung ◽  
Soluble Thrombomodulin ◽  
Molecular Weights ◽  
Dose Dependent Fashion ◽  
Dose Dependent

SummaryWe examined the anticoagulant activity of two major molecules of soluble thrombomodulin purified from human urine. The apparent molecular weights of these urinary thrombomodulins (UTMs) were 72,000 and 79,000, respectively. Both UTMs showed more potent cofactor activity for protein C activation [specific activity >5,000 thrombomodulin units (TMU)/mg] than human placental thrombomodulin (2,180 TMU/mg) and rabbit lung thrombomodulin (1,980 TMU/mg). The UTMs prolonged thrombin-induced fibrinogen clotting time (>1 TMU/ml), APTT (>5 TMU/ml), TT (>5 TMU/ml) and PT (>40 TMU/ml) in a dose-dependent fashion. These effects appeared in the concentration range of soluble thrombomodulins present in human plasma and urine. In the rat DIC model induced by thromboplastin, administration of UTMs by infusion (300-3,000 TMU/kg) restored the hematological abnormalities derived from DIC in a dose-dependent fashion. These results demonstrate that UTMs exhibit potent anticoagulant and antithrombotic activities, and could play a physiologically important role in microcirculation.

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