Activation of the inositol trisphosphate second messenger system by cAMP in a mouse fibroblast cell line

Intracellular Ca²⁺ mobilization events were assessed in mouse L cells, which contain native prostaglandin E₁ receptors and transfected human ₂ adrenergic receptors. Both Fura2 (single cell measurements) and Quin 2, (cuvette assays) were used to determine [Ca²⁺]_i levels. Our results demonstrate that in the transfected cells there is a dose-dependent increase in [Ca²⁺]_i in response to isoproterenol (0.1 nM–100 nM), which is inhibited by the -adrenergic antagonist, propranolol, and is a result of intracellular Ca²⁺ release. [Ca²⁺]₁ in these cells was also increased by prostaglandin E₁, 8 bromo cyclic AMP, and aluminum fluoride. Both 8 bromo cAMP and isoproterenol induced a rapid increase in the levels of IP₁, IP₂, and IP₃. The data presented demonstrate that the elevation of intracellular cyclic AMP induces an increase in IP₃ production which leads to an elevation in [Ca²⁺];. We propose that this cyclic AMP dependent activation of the IP₃ generating system occurs at a post-receptor site.Abbreviations cAMP Adenosine Cyclic 3-5-Monophosphate - [Ca²⁺]_i intracellular [Ca²⁺]_i - 8 Br cAMP 8 Bromo Adenosine Cyclic 3-5-Monophosphate - DAG Diacylglycerol - EGTA] [Ethylene Bis (oxyethylenenitrilo)] Tetracetic acid - BSA Bovine Serum Albumin - HBSS-H Hanks' Balanced Salt Solution buffered with HEPES to pH 7.4 - HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid - PIP₂ Phosphatidylinositol 4,5-bisphosphate - IP₂ Inositol 4 Phosphate - IP₂ Inositol 4,5 Bisphosphate - IP₃ Inositol Trisphosphate - PGE₁ Prostaglandin E₁ - PBS Phosphate Buffered Saline Solution     

Granulocyte-mediated airway edema in guinea pigs

To determine the role of polymorphonuclear leukocytes (PMNs) in the airway edema that accompanies airway inflammation, we studied the effects of a 1-h exposure to 2 ppm toluene diisocyanate (TDI) on tracheal extravasation of Evans blue dye and on the concentration of PMNs in the tracheal wall. Tracheal Evans blue content was significantly increased by TDI exposure (53.6 +/- 8.0 micrograms/g tracheal tissue (mean +/- SE) for animals exposed to TDI and 16.3 +/- 2.0 for animals exposed to air, P less than 0.0025) as were both the intravascular and extravascular concentration of PMNs in tracheal sections (intravascular PMNs were 28.0 +/- 8.4 X 10(3) cells/mm3 for TDI and 1.5 +/- 1.5 X 10(3) for air, P less than 0.025, extravascular PMNs were 10.9 +/- 4.5 X 10(3) for TDI and 0 for air, P less than 0.05). PMN depletion with vinblastine or with hydroxyurea abolished both the increase in tracheal Evans blue extravasation and the increase in the concentration of intravascular and extravascular PMNs in animals exposed to TDI. PMN depletion with hydroxyurea did not significantly inhibit the increase in tracheal Evans blue extravasation caused by intravenous histamine. Administration of donor PMNs to animals depleted of PMNs with hydroxyurea reconstituted the TDI-induced increase in tracheal Evans blue extravasation (80.4 +/- 17.3 micrograms/g tissue (mean +/- SE) in animals exposed to TDI vs. 21.3 +/- 2.9 in animals exposed to air, P less than 0.025) and in the intravascular concentration of PMNs in tracheal sections [18.5 +/- 3.4 X 10(3) cells/mm3 (mean +/- SE) in animals exposed to TDI vs. 1.3 +/- 1.3 X 10(3) in animals exposed to air, P less than 0.0025].(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of a factor from calf serum that promotes the pigmentation of embryonic and transformed melanocytes

A protein (Mr = 63,000) from calf serum that promotes the pigmentation of cultured chick neural crest and mouse melanoma cells has been partially isolated and characterized in this study. The stimulation of melanin synthesis in cultured cells was used to follow its activity during purification. The pigment-promoting factor was isolated by sequential column chromatography on dye-agarose matrices followed by hydroxyapatite and high pressure molecular sieve chromatography. The factor was found to stimulate melanin biosynthesis at 2-4 micrograms/ml and was specific for melanin-producing cells and their precursors. Antibodies raised in rabbits against the factor inhibited its pigment-promoting activity as well as that of whole calf serum. Enzyme-linked immunoadsorbent assays demonstrated that calf and bovine sera contain molecules that cross-react with the pigment-promoting factor. Horse, human, rat, and chicken sera, which lack the biological activity, also lacked immunological cross-reactivity. Extracts of certain tissues, particularly the submaxillary gland, were observed to be rich sources of pigment-promoting activity.     

Evidence that insulin and guanosine triphosphate regulate dephosphorylation of the beta-subunit of the insulin receptor in sarcolemma membranes isolated from skeletal muscle.

When sarcolemma membranes isolated from rat skeletal muscle were incubated with [gamma-32P]ATP, a membrane protein of apparent Mr 95,000 was rapidly phosphorylated, with the 32P content reaching a maximum within 2 s. On the basis of immunoprecipitation with anti-insulin-receptor antiserum, phosphoamino acid analysis and Mr, this protein probably represents the beta-subunit of the insulin receptor. Similarly, on incubation of the membrane with adenosine 5'-[gamma-[35S]thio] triphosphate the 95 kDa protein was thiophosphorylated, indicating thiophosphorylation of the beta-subunit of the insulin receptor on the basis of immunoprecipitation studies. The effect of insulin on the phosphorylation of this protein in the membrane was studied. Insulin induced a 20% decrease in the 32P labelling of the protein when the membranes were phosphorylated for 10 s. This insulin effect was dose-dependent, with half-maximal effect obtained at 2-3 nM-insulin. Addition of GTP, but not GDP or guanosine 5'-[beta, gamma-imido]triphosphate, enhanced the effect to 35% inhibition, with half-maximal effect of GTP obtained at 0.5 microM. GTP had no effect on the phosphorylation of the protein in the absence of insulin. Analysis of this insulin effect showed that insulin increased the rate of dephosphorylation of the 95 kDa protein in the membrane. In contrast, insulin had no effect on thiophosphorylation of the 95 kDa membrane protein after incubation with adenosine 5'-[gamma-[35S]thio]triphosphate. Since thiophosphorylated proteins are less sensitive to phosphatase action, these investigations suggest that insulin stimulated a protein phosphatase activity in a GTP-dependent manner. The possibility that GTP-regulatory proteins are involved in the action of insulin on the phosphorylation of the insulin receptor and other membrane proteins is discussed.     

Statistic evaluation of the influence of species variation,culture conditions,surface wettability and fluid shear on attachment and biofilm development of marine bacteria

A budding coccoid bacterium, (CH1), a Vibrio sp. and a Pseudomonas sp. were investigated for factors governing their attachment to glass surfaces in static batch culture and laminar flow continuous culture systems. An analysis of variance showed that the three species exhibited very different responses. For CH1 attachment was dependent on cell density, incubation time and nutrient concentration. The Vibrio sp. was affected by nutrient concentration while the attachment of the Pseudomonas sp. was independent of cell density, incubation time and nutrient concentration. A comparison of attachment to hydrophilic and hydrophobic surfaces showed that attachment of the Vibrio sp. and CH1 to hydrophilic surfaces was 3 and 10 times greater respectively than to hydrophobic surfaces while Pseudomonas attached in equal numbers to both surfaces. The continuous culture system with defined flow hydrodynamics and growth conditions at steady state revealed a random sampling effect 3 times smaller than the batch culture system did. When the biofilm development of Pseudomonas sp. was followed during 46 h at different fluid shear under laminar and turbulent flow conditions, the former biofilm reached 3.3·10⁸ cells·cm^-2 and the latter 8.2·10⁷ cells·cm^-2.Non-common abbreviation NSS Nine salt solution     

In vivo dopamine receptor binding studies with a non-radioactively labeled agonist,dipropyl-5, 6-ADTN

Evidence is presented that a low dose of peripherally administered N, N-dipropylamino-5, 6-dihydroxytetralin (DiPr-5, 6-ADTN) specifically labels dopamine (DA) receptors in rat brain.Concentrations of this potent DA receptor agonist were determined by a highly selective method using reversed phase liquid chromatography and amperometric detection. The binding characteristics satisfy all criteria regarding saturability, stereospecificity, regional distribution and relation with pharmacological effects that are associated with DA receptor interactions. A rough estimation of the density of binding sites in the striatum resulted in values of 60–70 pmol/g. Lesioning the nigrostriatal pathway does not significantly alter the amount of ligand bound, nor do pretreatments with serotonergic, α-adrenergic or β-adrenergic antagonists. DiPr-5, 6-ADTN has thus been shown to be a useful ligand for labeling central DA receptors and a powerful tool in the study of DA-ergic mechanisms.