scholarly journals Classification of prostaglandin receptors based on coupling to signal transduction systems

1989 ◽  
Vol 263 (3) ◽  
pp. 769-774 ◽  
Author(s):  
S Muallem ◽  
B S Merritt ◽  
J Green ◽  
C R Kleeman ◽  
D T Yamaguchi
Keyword(s):  
Signal Transduction ◽  
Cyclic Amp ◽  
Wide Spectrum ◽  
Second Messengers ◽  
Osteosarcoma Cell ◽  
Prostaglandin Receptors ◽  
Umr 106 ◽  
Stimulation Of ◽  
Signal Transduction Systems

A wide spectrum of prostaglandins (PG) stimulate both the production of cyclic AMP and an increase in free cytosolic Ca2+ concentration [(Ca2+]i) in the osteogenic osteosarcoma cell line, UMR-106-01, which has characteristics compatible with osteoblasts. Using PG-stimulated determinations of the second messengers cyclic AMP and [Ca2+]i, a method for classification of PG receptors is presented. UMR-106-01 cells demonstrate three subclasses of PG receptors. One receptor interacts with PGF2 alpha, PGD2, and thromboxane B2 (TxB2) to increase [Ca2+]i. A second receptor binds PGE2, PGE1, PGI2, PGA2 and 6-oxo-PGF1 alpha to increase [Ca2+]i by stimulation of a second separate phospholipase C pool. A third receptor accepts PGE2, PGE1, PGA2, PGI2 and to a lesser extent PGF2 alpha, PGD2 and TxB2 to increase cyclic AMP. Such a classification system may be applicable to other cells responding to multiple PGs by inducing changes in cellular second messengers.

scholarly journals Use of phosphosynapsin I-specific antibodies for image analysis of signal transduction in single nerve terminals

2000 ◽  
Vol 113 (20) ◽  
pp. 3573-3582 ◽  
Author(s):  
A. Menegon ◽  
D.D. Dunlap ◽  
F. Castano ◽  
F. Benfenati ◽  
A.J. Czernik ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Nerve Terminal ◽  
Polyclonal Antibodies ◽  
Second Messengers ◽  
Nerve Terminals ◽  
Dependent Protein Kinase ◽  
Spatially Resolved ◽  
Dependent Protein

We have developed a semi-quantitative method for indirectly revealing variations in the concentration of second messengers (Ca(2+), cyclic AMP) in single presynaptic boutons by detecting the phosphorylation of the synapsins, excellent nerve terminal substrates for cyclic AMP- and Ca(2+)/calmodulin-dependent protein kinases. For this purpose, we employed polyclonal, antipeptide antibodies recognising exclusively synapsin I phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II (at site 3) or synapsins I/II phosphorylated by either cAMP-dependent protein kinase or Ca(2+)/calmodulin-dependent protein kinase I (at site 1). Cerebellar granular neurones in culture were double-labelled with a monoclonal antibody to synapsins I/II and either of the polyclonal antibodies. Digitised images were analysed to determine the relative phosphorylation stoichiometry at each individual nerve terminal. We have found that: (i) under basal conditions, phosphorylation of site 3 was undetectable, whereas site 1 exhibited some degree of constitutive phosphorylation; (ii) depolarisation in the presence of extracellular Ca(2+) was followed by a selective and widespread increase in site 3 phosphorylation, although the relative phosphorylation stoichiometry varied among individual terminals; and (iii) phosphorylation of site 1 was increased by stimulation of cyclic AMP-dependent protein kinase but not by depolarisation and often occurred in specific nerve terminal sub-populations aligned along axon branches. In addition to shedding light on the regulation of synapsin phosphorylation in living nerve terminals, this approach permits the spatially-resolved analysis of the activation of signal transduction pathways in the presynaptic compartment, which is usually too small to be studied with other currently available techniques.

scholarly journals Extracellular ATP stimulates three different receptor-signal transduction systems in FRTL-5 thyroid cells. Activation of phospholipase C, and inhibition and activation of adenylate cyclase

1992 ◽  
Vol 283 (1) ◽  
pp. 281-287 ◽  
Author(s):  
K Sato ◽  
F Okajima ◽  
Y Kondo
Keyword(s):  
Signal Transduction ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Phospholipase C ◽  
Pertussis Toxin ◽  
Extracellular Atp ◽  
Thyroid Cells ◽  
Receptor Signal ◽  
Atp Analogues ◽  
Signal Transduction Systems

In FRTL-5 thyroid cells, extracellular ATP, a P2-agonist, not only stimulates phospholipase C but also inhibits forskolin- or thyrotropin (TSH)-induced stimulation of adenylate cyclase in a pertussis toxin-sensitive manner [Okajima, Sato, Nazarea, Sho, & Kondo (1989) J. Biol. Chem. 264, 13029-13037]. We have now found that, in pertussis toxin-treated cells, ATP can directly stimulate adenylate cyclase. Although adenylate cyclase modulation occurs through ATP metabolites such as AMP and adenosine, we show that extracellular ATP itself also regulates cyclic AMP production, based on the following: (1) the actions of ATP were imitated by hydrolysis-resistant ATP analogues, (2) the elimination of adenosine by adenosine deaminase decreased the effect of ATP only partially, at least at concentrations greater than 10 microM-ATP, and (3) the amount of AMP produced from ATP was too low to account for the ATP effects. To identify the respective receptors for the three different actions of ATP, we established an antagonist profile. Suramin, which has been reported to be a P2-receptor antagonist, inhibited ATP-induced phospholipase C activation in a competitive fashion, but did not affect ATP-induced adenylate cyclase modulation. On the other hand, 8-cyclopentyl-1,3-diphenylxanthine competitively antagonized both the stimulatory and inhibitory ATP actions on cyclic AMP levels, but did not influence the activation of phospholipase C by ATP. The order of potency for various xanthine derivatives was clearly different with respect to their antagonistic effects on the stimulation and inhibition of adenylate cyclase induced by ATP. We conclude that ATP activates three receptors, each of which is coupled to a different signal transduction system in FRTL-5 cells, i.e. phospholipase C activation, and adenylate cyclase activation and inhibition.

Effects of parathyroid hormone on cyclic AMP-formation and cytoplasmic free Ca2+ in the osteosarcoma cell line UMR 106-01

1992 ◽  
Vol 12 (3) ◽  
pp. 207-214 ◽  
Author(s):  
Östen Ljunggren ◽  
Hans Johansson ◽  
Ulf H. Lerner ◽  
Erik Lindh ◽  
Sverker Ljunghall
Keyword(s):  
Parathyroid Hormone ◽  
Cyclic Amp ◽  
Cell Line ◽  
Single Cells ◽  
Transient Increase ◽  
Osteosarcoma Cell Line ◽  
Osteosarcoma Cell ◽  
Fura 2 ◽  
Umr 106 ◽  
Camp Formation

The effects of parathyroid hormone (PTH) on cytoplasmic free CA2+ (Cai2+) and cAMP-formation were investigated in the rat osteosarcoma cell line UMR 106-01. In fura-2 loaded adherent single cells bPTH 1–34 (10 nM−1μM) induced a rapid transient increase in Cai2+ in 11% of the studied cells. In fura-2 tracings from UMR 106-01 cells in suspension, bPTH 1–34 (0.1 μM) induced a transient increase in Cai2+ in 20% of the experiments. The transient increase in Cai2+ seen in suspensions of cells was not abolished by addition of EGTA (2.5 mM) prior to challenge with PTH, suggesting that the increase in Cai2+ was derived from intracellular stores. A marked rapid increase in cAMP-formation was observed in all experiments with cells in suspension, also in the experiments where PTH did not affect Cai2+. These data show that PTH causes a release of Ca2+ from intracellular stores in a small percentage of osteosarcoma UMR 106-01 cells, and that PTH is capable of inducing an increase in cAMP-formation without affecting Cai2+ in osteoblasts.

Regulation of Na-K-2Cl cotransport in osteoblasts

1991 ◽  
Vol 261 (3) ◽  
pp. C433-C440 ◽  
Author(s):  
N. Whisenant ◽  
B. X. Zhang ◽  
M. Khademazad ◽  
P. Loessberg ◽  
S. Muallem
Keyword(s):  
Osteosarcoma Cell ◽  
Dependent Manner ◽  
Hypotonic Medium ◽  
Cell Shrinkage ◽  
Concentration Dependent Manner ◽  
Kinase C ◽  
Maximal Stimulation ◽  
Cotransport System ◽  
Umr 106 ◽  
Stimulation Of

Uptake of 86Rb was used to follow the activity of Na-K-2Cl cotransport in the osteosarcoma cell line UMR-106-01. The ouabain-resistant fraction of 86Rb uptake was sensitive to bumetanide and furosemide. Furosemide-sensitive 86Rb uptake required the presence of Na+, K+, and Cl- in the incubation medium. These observations indicate the presence of a Na-K-2Cl cotransport system in osteoblasts. Cotransporter activity was stimulated by agonists which increase adenosine 3',5'-cyclic monophosphate (cAMP), cytosolic free Ca2+ ([Ca2+]i), and protein kinase C (PKC) activity such as parathyroid hormone (PTH) and prostaglandin E2 (PGE2). However, endothelin, which increases [Ca2+]i and PKC activity without affecting cellular levels of cAMP, was ineffective in stimulating the cotransporter. Accordingly, increasing cellular cAMP with forskolin was as effective as PTH and PGE2 in stimulating the cotransporter. Stimulation of PKC with TPA inhibited the cotransporter in a time- and concentration-dependent manner. No stimulation of cotransport could be demonstrated at any 12-O-tetradecanoyl-phorbol-13-acetate (TPA) concentration or incubation time. The Na-K-2Cl cotransporter was stimulated by cell shrinkage. Maximal stimulation was observed after swelling the cells in hypotonic medium and subsequent shrinkage in isotonic medium. Stimulation by cell shrinkage can be demonstrated in control, agonist-, cAMP-, and TPA-treated cells. These observations suggest that 1) the osteoblastic Na-K-2Cl cotransporter is activated by calciotropic hormones predominantly through an increase in cellular cAMP, and 2) in osteoblasts, the cotransporter is independently regulated by different biochemical pathways.

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