scholarly journals Muscarinic-agonist and guanine nucleotide activation of polyphosphoinositide phosphodiesterase in isolated islet-cell membranes

1986 ◽  
Vol 240 (3) ◽  
pp. 731-737 ◽  
Author(s):  
M E Dunlop ◽  
R G Larkins
Keyword(s):  
Islet Cell ◽  
Plasma Membranes ◽  
Islet Cells ◽  
Muscarinic Agonist ◽  
Dependent Manner ◽  
Muscarinic Agonists ◽  
Phospholipid Hydrolysis ◽  
Gtp Binding ◽  
Inositol Phospholipids ◽  
Hydrolysis Of

Stimulated hydrolysis of the inositol phospholipids phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] was investigated by studying the phosphoinositides produced in a suspended preparation of plasma membranes by transference of 32P from [gamma-32P]ATP. At basal Ca2+ concentration (calculated free Ca2+, 150 nM) phospholipid hydrolysis was stimulated either by the muscarinic agonists carbamoylcholine and bethanecol or by the addition of the non-hydrolysable analogue of GTP, guanosine 5′-[beta gamma-imido]triphosphate [p(NH)ppG]. GTP was without effect on basal hyrolysis. Both GTP and p(NH)ppG enhanced the rapid (within 10 s) hydrolysis of PtdIns4P and PtdIns(4,5)P2 induced by carbamoylcholine in a dose-dependent manner. A rightward shift in the competition curve of carbamoylcholine for bound L-[3H]quinuclidinyl benzilate was seen on addition of GTP or p(NH)ppG (100 microM) under phosphorylating conditions. Pretreatment of intact islet cells with Bordetella pertussis toxin, islet-activating protein (IAP) or treatment of membranes with IAP under conditions which elicited ADP-ribosylation of a protein of Mr 41,000 was without effect on muscarinic binding, phosphoinositide phosphorylation or subsequent hydrolysis by carbamoylcholine. The findings indicate the involvement of a GTP-binding protein in the coupling of the muscarinic receptor to phosphoinositide hydrolysis in the islet cell and suggest that this is distinct from the GTP-binding regulatory component of adenylate cyclase which is covalently modified by IAP.

scholarly journals Alloxan cytotoxicity in vitro. Inhibition of rubidium ion pumping in pancreatic β-cells

1977 ◽  
Vol 162 (1) ◽  
pp. 9-18 ◽  
Author(s):  
L Å Idahl ◽  
Å Lernmark ◽  
J Sehlin ◽  
I B Täljedal
Keyword(s):  
Plasma Membranes ◽  
Islet Cells ◽  
Protective Action ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Nitrophenyl Phosphate ◽  
Cation Pump ◽  
Ion Pumping ◽  
Hydrolysis Of

Exposing micro-dissected pancreatic islets of non-inbred ob/ob mice to 2-5 mM-alloxan for 10 min decreased the ability of the islets to accumulate Rb+. Rb+ accumulation in pieces of exocrine pancreas was unaffected by alloxan. When islets were treated with alloxan in the presence of 2-20 mM-D-glucose, the Rb+-accumulating ability was protected in a dose-dependent manner. The protective action of D-glucose was reproduced with 3-O-methyl-D-glucose but not with L-glucose or D-mannoheptulose; mannoheptulose prevented D-glucose from exerting its protective action. The inhibition of Rb+ accumulation was due to a decreased inward pumping, since alloxan did not affect Rb+ efflux from pre-loaded islets. The inhibitory effect of alloxan had a latency of about 1 min, as revealed by experiments with dispersed islet cells in suspension. Alloxan-treated islets showed only a marginal decrease in ATP and no change in glucose 6-phosphate concentration. Although alloxan slightly decreased the hydrolysis of ATP in a subcellular fraction enriched in plasma membranes, this effect could not be attributed to a ouabain-sensitive adenosine triphosphatase. The plasma membranes exhibited a K+-activated hydrolysis of p-nitrophenyl phosphate; this enzyme activity too was insensitive to alloxan. Glucose may protect the univalent-cation pump by preventing permeation of alloxan via a path coupled to the hexose-transport system. Inhibition of the pump may be fundamental to the induction of alloxan-diabetes.

scholarly journals The roles of phospholipase D and a GTP-binding protein in guanosine 5′-[γ-thio]triphosphate-stimulated hydrolysis of phosphatidylcholine in rat liver plasma membranes

1990 ◽  
Vol 272 (3) ◽  
pp. 749-753 ◽  
Author(s):  
K M Hurst ◽  
B P Hughes ◽  
G J Barritt
Keyword(s):  
Rat Liver ◽  
Phospholipase D ◽  
Plasma Membranes ◽  
Regulatory Protein ◽  
Gtp Binding ◽  
Liver Plasma Membranes ◽  
Low Concentrations ◽  
Rat Liver Plasma Membranes ◽  
Triton X ◽  
Hydrolysis Of

1. Guanosine 5′-[gamma-thio]triphosphate (GTP[S]) stimulated by 50% the rate of release of [3H]choline and [3H]phosphorylcholine in rat liver plasma membranes labelled with [3H]choline. About 70% of the radioactivity released in the presence of GTP[S] was [3H]choline and 30% was [3H]phosphorylcholine. 2. The hydrolysis of phosphorylcholine to choline and the conversion of choline to phosphorylcholine did not contribute to the formation of [3H]choline and [3H]phosphorylcholine respectively. 3. The release of [3H]choline from membranes was inhibited by low concentrations of SDS or Triton X-100. Considerably higher concentrations of the detergents were required to inhibit the release of [3H]phosphorylcholine. 4. Guanosine 5′-[beta gamma-imido]triphosphate and guanosine 5′-[alpha beta-methylene]triphosphate, but not adenosine 5′-[gamma-thio]-triphosphate, stimulated [3H]choline release to the same extent as did GTP[S]. The GTP[S]-stimulated [3H]choline release was inhibited by guanosine 5′-[beta-thio]diphosphate, GDP and GTP but not by GMP. 5. It is concluded that, in rat liver plasma membranes, (a) GTP[S]-stimulated hydrolysis of phosphatidylcholine is catalysed predominantly by phospholipase D with some contribution from phospholipase C, and (b) the stimulation of phosphatidylcholine hydrolysis by GTP[s] occurs via a GTP-binding regulatory protein.

scholarly journals Chlorotetracycline as a fluorescent Ca2+ probe in pancreatic islet cells.

1978 ◽  
Vol 76 (3) ◽  
pp. 652-674 ◽  
Author(s):  
I B Täljedal
Keyword(s):  
Plasma Membranes ◽  
Islet Cells ◽  
Dependent Manner ◽  
Cell Surfaces ◽  
Noticeable Effect ◽  
Cell Plasma ◽  
Glass Capillaries ◽  
Asymmetrically Distributed ◽  
Dose Dependent ◽  
Dispersed Cells

Pancreatic islets, or suspensions of islet cells, from noninbred ob/ob-mice were incubated with chlorotetracycline and analyzed for Ca2+-dependent fluorescence in a microscope. Unless logarithmically transformed, signals from islets were asymmetrically distributed with unstable variance. Signals from cells pelleted in glass capillaries were more homogeneous and depended linearly on the thickness of the sample. The effect of sample thickness and a significant enhancement of fluorescence by alloxan suggest that beta-cells were involved in producing the signal from whole islets. The signal from dispersed cells was probably diagnostic of Ca2+ in beta-cell plasma membranes because it was suppressed by La3+ and had a spectrum indicative of an apolar micromilieu; fluorescent staining of cell surfaces was directly seen at high magnification. Fluorescence from cells was enhanced by 0.5-10 mM Ca2+ in a dose-dependent manner, whereas less than 0.5 mM Ca2+ saturated the probe alone in methanol. The signal from islets or dispersed cells was suppressed by 5 mM theophylline; that from cells was also suppressed by 0.5 mM 3-isobutyl-1-methylxanthine, 1.2 or 15 mM Mg2+, 3-20 mM D-glucose, and, to a lesser extent, 20 mM 3-O-methyl-D-glucose. D-glucose was more inhibitory in the absence than in the presence of Mg2+, as if Mg2+ and D-glucose influenced the same Ca2+ pool. L-glucose, D-mannopheptulose, or diazoxide had no noticeable effect and 20 mM bicarbonate was stimulatory. The results suggest that microscopy of chlorotetracycline-stained cells can aid in characterizing calcium pools of importance for secretion. Initiation of insulin release may be associated with an increas

Light-induced interaction between rhodopsin and GTP-binding protein leads to the hydrolysis of GTP in the rod outer segment

1986 ◽  
Vol 64 (4) ◽  
pp. 304-308 ◽  
Author(s):  
B. D. Gupta ◽  
T. J. Borys ◽  
S. Deshpande ◽  
R. E. Jones ◽  
E. W. Abrahamson
Keyword(s):  
Light Scattering ◽  
Outer Segment ◽  
Plasma Membranes ◽  
Binding Protein ◽  
Rod Outer Segments ◽  
Particle Scattering ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Time Period ◽  
Hydrolysis Of

In the presence of exogeneous GTP, vertebrate whole rod outer segments (ROS), with perforated plasma membranes in the "single particle" scattering range, elicit a light-induced light-scattering transient which we call the "G" signal. Here, we report on the characteristics of the "G" signal relative to the "binding" and "dissociation" signals reported by Kuhn and colleagues. Replacing GTP with guanylyl imidodiphosphate (GMP-PNP) does not give rise to the G signal. This indicates that hydrolysis of the terminal phosphate is required for the G signal and, in addition. GTP and GMP-PNP compete for the same binding site of the enzyme responsible for the G signal (i.e., GTP-binding protein). Also, neither GDP nor its nonhydrolyzable analogue, guanosine 5′-O-(2-thiodiphosphate), when present in ROS suspensions yield any light-scattering transient in the time period tested.

scholarly journals Insulin activates GTP binding to a 40 kDa protein in fat cells

1991 ◽  
Vol 276 (1) ◽  
pp. 103-108 ◽  
Author(s):  
M Kellerer ◽  
B Obermaier-Kusser ◽  
A Pröfrock ◽  
E Schleicher ◽  
E Seffer ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Insulin Receptor ◽  
Binding Site ◽  
G Protein ◽  
Plasma Membranes ◽  
Fat Cells ◽  
Dependent Manner ◽  
Receptor Kinase ◽  
Gtp Binding ◽  
Insulin Receptor Kinase

The first steps in insulin action are binding of insulin to its receptor and activation of the insulin receptor kinase. As there is indirect evidence that further signal transduction might involve a guanine-nucleotide-binding protein (G-protein), we studied whether insulin modulates GTP binding to plasma membrane proteins of fat cells and skeletal muscle. We found that insulin rapidly increased (30 s) binding of guanosine 5′-[gamma-thio]triphosphate (GTP[S]) in a dose dependent manner (0.03-2.0 nM). This effect was not altered by pertussis toxin, but it was abolished by cholera toxin treatment of fat cells. Scatchard analysis of the binding data showed that the increased GTP[S] binding is due to a decrease in the Kd for GTP from 100 nM to 50 nM. Furthermore, binding of GTP to these plasma membranes inhibited both the binding of 125I-insulin to the insulin receptor and the stimulation of the insulin receptor kinase, suggesting a feedback interaction between the insulin-stimulated GTP-binding site and the insulin receptor. In order to identify this insulin-stimulated GTP-binding site, plasma membranes were labelled with the photoreactive GTP analogue [alpha-32P]GTP gamma-azidoanilide. We found that insulin selectively stimulated GTP binding to a 40 kDa protein. In conclusion, in plasma membranes of fat cells and skeletal muscle, the insulin receptor interacts with a 40 kDa GTP-binding site. We speculate that this 40 kDa GTP-binding site might be a G-protein which is involved in insulin signal transmission.

scholarly journals Quantification of inositol phospholipid breakdown in isolated rat hepatocytes

1993 ◽  
Vol 290 (3) ◽  
pp. 865-872 ◽  
Author(s):  
C J Allan ◽  
J H Exton
Keyword(s):  
Phospholipase C ◽  
Phospholipase D ◽  
Inositol Phosphates ◽  
Rat Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Phospholipid Hydrolysis ◽  
Inositol Phospholipids ◽  
Inositol Phospholipid ◽  
Inositol Phospholipid Breakdown ◽  
Hydrolysis Of

The hydrolysis of inositol phospholipids induced by vasopressin in hepatocytes during 60 min was quantified chemically. There was a large release of myo-inositol which was abolished by Li+, indicating that it was derived from inositol phosphates and not from phospholipase D action on PtdIns. There was also a large release of inositol phosphates which was increased approx. 2-fold by Li+ at 30 min, but then remained constant, suggesting that inositol phospholipid breakdown declined substantially beyond this time. In cells prelabelled with myo-[3H]inositol and treated with Li+, [3H]PtdIns(4,5)P2 decreased maximally (50%) at 15 s and then recovered to a level at 5 min that was maintained at 25% below control for 40 min. [3H]PtdIns4P and [3H]PtdIns showed slower decreases to approx. 30% below control at 15 min, but with no further changes. Labelled Ins(1,4,5)P3 and Ins(1,3,4)P3 showed 2-4-fold increases within 30 s and then declined to values that were maintained at a constant level above the control, except for [3H]Ins(1,3,4)P3, which showed a second increase. [3H]Ins(1,4)P2 showed a very large increase over 10 min, whereas [3H]Ins4P and [3H]Ins1P showed little change before 6 and 15 min respectively. The total [3H]inositol phosphates showed little further increase after 20 min. These data are consistent with a rapid, but not sustained, hydrolysis of PtdIns-(4,5)P2, but not of PtdIns, by phospholipase C, but do not exclude PtdIns4P as a substrate. Phosphatidate was rapidly increased by vasopressin, whereas diacylglycerol was increased after a 1-2 min lag. Both were maintained at levels 2-3-fold above control for 60 min. The vasopressin-induced increase in inositol phosphates plus myo-inositol (approx. 120 nmol/100 mg) was greater than the increase in diacylglycerol plus phosphatidate (approx. 60 nmol/100 mg) between 10 and 40 min. This indicates that there was substantial further metabolism of these lipids. Addition of 75 mM ethanol resulted in rapid production of phosphatidylethanol in response to vasopressin and a 35% reduction in phosphatidate, but no decrease in diacylglycerol. In summary, the results indicate that inositol phospholipid hydrolysis by phospholipase C can account for most of the diacylglycerol and phosphatidate that accumulate during 60 min of vasopressin action, but that these phospholipids are probably not the major source of the phosphatidate that is formed during the first 2 min by phospholipase D, or of the diacylglycerol and phosphatidate that are formed beyond 30 min.

THERAPIE EINES METASTASIERENDEN INSELZELLTUMORS MIT STREPTOZOTOCIN

1971 ◽  
Vol 67 (2) ◽  
pp. 405-416 ◽  
Author(s):  
E. Nieschlag ◽  
H. Wombacher ◽  
F. J. Kroeger ◽  
L.V. Habighorst
Keyword(s):  
Liver Metastases ◽  
Blood Sugar ◽  
Mechanism Of Action ◽  
Islet Cell ◽  
Plasma Insulin ◽  
Severe Hypoglycaemia ◽  
Islet Cells ◽  
Intravenous Injections ◽  
High Affinity ◽  
Methyl Nitrosourea

A patient with a metastazing functional islet cell tumour suffering from severe hypoglycaemia was treated with streptozotocin. Four intravenous injections of 1.5 g streptozotocin each were administered in 4 to 6 days intervals. After the 4th injection there were no further episodes of hypoglycaemia, parenteral glucose administration could be stopped and blood sugar and plasma insulin, showing concentrations of up to 405 μU/ml before treatment, reached normal levels. The tumours in the pancreas disappeared and the liver metastases decreased in size and number as judged by arteriography. A hypothesis for the mechanism of action of streptozotocin is proposed. The glucose moiety is considered to facilitate a high affinity to the islet cells whereas the N-methyl-nitrosourea residue serves the active antitumour part of the molecule.

scholarly journals Immunohistochemistry of Pancreatic Islet Cell Tumors in the Ferret (Mustela putorius furo)

1997 ◽  
Vol 34 (5) ◽  
pp. 387-393 ◽  
Author(s):  
G. A. Andrews ◽  
N. C. Myers ◽  
C. Chard-Bergstrom
Keyword(s):  
Pancreatic Islets ◽  
Pancreatic Islet ◽  
Pancreatic Polypeptide ◽  
Islet Cell ◽  
Islet Cells ◽  
Neuron Specific Enolase ◽  
Islet Cell Tumors ◽  
Pancreatic Islet Cell ◽  
Formalin Fixed Paraffin Embedded ◽  
Pancreatic Islet Cell Tumors

Twenty-two pancreatic islet cell tumors and normal pancreatic islets from ferrets were evaluated by immunohistochemistry for expression of the peptide hormones insulin, somatostatin, glucagon, and pancreatic polypeptide (PP) and the neuroendocrine markers chromogranin A (CgA) and neuron-specific enolase (NSE). In normal pancreatic islets, the majority of cells stained strongly with CgA and NSE. A cells, B cells, D cells, and PP cells stained strongly with glucagon, insulin, somatostatin, and PP, respectively. All 22 tumors stained with CgA and NSE. The proportion of cells within tumors staining for CgA was variable, but more than half of the cells stained positively in 18 of the tumors. The intensity of staining for CgA was strong (reactivity equivalent to or greater than normal islet cells in adjacent tissue) in 11 moderate in six, and weak in five of the tumors. All tumors stained for NSE, with ≥50% of the cells staining in 21 of the tumors, and the intensity of staining was strong in 18 of the tumors. Twenty of 22 tumors stained positively for insulin, with ≥50% of the cells staining in 19 of them. The intensity of staining for insulin was strong in 12, moderate in seven, and weak in one of the tumors. Approximately ≤1% of the cells in 15 of 22 tumors stained for somatostatin, five tumors stained for pancreatic polypeptide, and three tumors stained for glucagon. These data indicate that the majority of islet cell tumors of ferrets express immunohistochemically detectable insulin. CgA and NSE are both useful general markers for such tumors, including those that are insulin negative. Commercially available antisera to CgA, NSE, insulin, glucagon, somatostatin, and PP work well in formalin-fixed, paraffin-embedded tissue for immunophenotyping islet cell tumors in the ferret.

scholarly journals Low-density-lipoprotein-receptor-related protein (LRP) interacts with a GTP-binding protein

1998 ◽  
Vol 336 (2) ◽  
pp. 381-386 ◽  
Author(s):  
Lothar GORETZKI ◽  
Barbara M. MUELLER
Keyword(s):  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Low Density ◽  
Lipoprotein Receptor ◽  
Dependent Manner ◽  
Related Protein ◽  
Gtp Binding ◽  
Density Lipoprotein Receptor ◽  
Dose Dependent ◽  
Lipoprotein Receptor Related Protein

The low-density-lipoprotein-receptor-related protein (LRP) binds and internalizes numerous ligands, including lipoproteins, proteinase–inhibitor complexes and others. We have shown previously that LRP-mediated ligand internalization is dependent on cAMP-dependent protein kinase (PKA) activity. Here, we investigated whether ligation of LRP increases the intracellular cAMP level and PKA activity via a stimulatory GTP-binding protein. Treatment of LRP-expressing cell lines with the LRP ligands lactoferrin or urokinase-type plasminogen activator caused a significant elevation in cAMP and stimulated PKA activity in a dose-dependent manner. Addition of the 39 kDa receptor-associated protein (RAP), an antagonist for ligand interactions with LRP, blocked the lactoferrin-induced increase in PKA activity, demonstrating a requirement for ligand binding to LRP. Incubation of cell membrane fractions with lactoferrin increased GTPase activity in a time- and dose-dependent manner, and treatment with LRP ligands suppressed cholera-toxin-mediated ADP-ribosylation of the Gsα subunit of a heterotrimeric G-protein. Affinity precipitation of LRP with RAP resulted in co-precipitation of two isoforms of Gsα from detergent extracts. We thus conclude that LRP is a signalling receptor that associates directly with a stimulatory heterotrimeric G-protein and activates a downstream PKA-dependent pathway.

Detection of islet cell surface antibodies by an indirect rosette assay, using islet cells and protein A-labeled sheep red blood cells

1984 ◽  
Vol 74 (1) ◽  
pp. 173-180 ◽  
Author(s):  
Shoji Maruyama ◽  
Masahiko Sugiura ◽  
Michio Nakazawa ◽  
Hiroko Tomiyama ◽  
Miyuki Shizawa ◽  
...  
Keyword(s):  
Cell Surface ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Islet Cell ◽  
Islet Cells ◽  
Protein A ◽  
Sheep Red Blood Cells ◽  
Islet Cell Surface Antibodies ◽  
Surface Antibodies
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