calcimycin and 3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate

15-Hydroxyeicosatetraenoic acid [15-(S)-HETE], a major arachidonic acid metabolite produced from the 15-lipoxygenase pathway, has been characterized as an antiinflammatory cellular mediator since it can inhibit the in vivo and in vitro formation of the proinflammatory leukotrienes via the 5-lipoxygenase pathway in various cells. 15-HETE has been confirmed to inhibit the 5-lipoxygenase in rat basophilic leukemia cell (RBL-1) homogenates with an I50 = 7.7 microM. The I50 of the 12-HETE isomer was 6 microM whereas prostaglandin F2 alpha was ineffective. In order to examine the mechanistic basis underlying the inhibitory action of 15-HETE, association assays of [3H]-15-HETE with RBL-1 subcellular fractions were carried out. The presence of the zwitterionic detergent CHAPS enhanced specific [3H]-15-HETE binding in the membrane fractions three-fold and specific 15-HETE binding was distributed among the nuclear (32%)-, granule (19%)-, plasma membrane (35%)-, and cytosol (14%)-enriched fractions. Studies using combined granule and plasma membrane enriched-, CHAPS treated-fractions showed that [3H]-15-HETE binding was time-dependent, specific and reversible, sensitive to pertussis toxin treatment, and indicated a single class of binding sites with a Kd = 460 +/- 160 nM and Bmax = 5.0 +/- 1.1 nM. Competition experiments showed that the order of 15-HETE or analogs in inhibiting the binding of [3H]-15-HETE was: 15(S)-HETE > or = 12-(S)-HETE = 5-(S)-HETE > 15-(R)-HETE > arachidonic acid. Prostaglandin F2 alpha and lipoxin B4 were ineffective as competitors. The similar profiles of the binding assays and inhibition of the 5-lipoxygenase suggest that 15-HETE binding sites may mediate this inhibitory action of 15-HETE.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Binding Sites; Binding, Competitive; Calcimycin; Cell Line; Cholic Acids; Dinoprost; Hydroxyeicosatetraenoic Acids; Leukemia, Basophilic, Acute; Lipoxygenase Inhibitors; Rats; Subcellular Fractions; Tumor Cells, Cultured

Mediatophore is a nerve terminal membrane protein purified from Torpedo electric organ on its ability to translocate acetylcholine upon calcium action. An antiserum able to immunoprecipitate mediatophore activity was used to study the subcellular distribution of this protein. The presynaptic membrane exhibited a strong and discontinuous immunogold labelling, especially at the active zone where ACh is thought to be released. Two antigens were recognized on immunoblots of synaptosomal membranes: the 15-kDa subunit of mediatophore and a 14-kDa membrane protein that has a wide non-neuronal distribution. Antibodies purified from the serum on native mediatophore and monospecific towards the 15-kDa antigen still gave a high presynaptic membrane localized labelling. In addition, a few 14-kDa protein sites were present at the active zone. The Schwann cell finger interposed between the presynaptic membrane and the postsynaptic arch also exhibited the 14-kDa antigen raising the question of a possible interaction of mediatophore with the 14-kDa protein originating from the Schwann cell.

Topics: Acetylcholine; Animals; Calcimycin; Calcium; Cholic Acids; Electric Organ; Microscopy, Immunoelectron; Molecular Weight; Nerve Endings; Nerve Tissue Proteins; Synaptosomes; Torpedo

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), a second messenger molecule involved in actions of neurotransmitters, hormones and growth factors, releases calcium from vesicular non-mitochondrial intracellular stores. An Ins(1,4,5)P3 binding protein, purified from brain membranes, has been shown to be phosphorylated by cyclic-AMP-dependent protein kinase and localized by immunohistochemical techniques to intracellular particles associated with the endoplasmic reticulum. Although the specificity of the Ins(1,4,5)P3 binding protein for inositol phosphates and the high affinity of the protein for Ins(1,4,5)P3 indicate that it is a physiological Ins(1,4,5)P3 receptor mediating calcium release, direct evidence for this has been difficult to obtain. Also, it is unclear whether a single protein mediates both the recognition of Ins(1,4,5)P3 and calcium transport or whether these two functions involve two or more distinct proteins. In the present study we report reconstitution of the purified Ins(1,4,5)P3 binding protein into lipid vesicles. We show that Ins(1,4,5)P3 and other inositol phosphates stimulate calcium flux in the reconstituted vesicles with potencies and specificities that match the calcium releasing actions of Ins(1,4,5)P3. These results indicate that the purified Ins(1,4,5)P3 binding protein is a physiological receptor responsible for calcium release.

Topics: Animals; Binding, Competitive; Brain; Calcimycin; Calcium; Calcium Channels; Cholic Acids; Chromatography, Affinity; Detergents; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Kinetics; Liposomes; Membranes; Molecular Weight; Phosphatidylcholines; Receptors, Cell Surface; Receptors, Cytoplasmic and Nuclear; Second Messenger Systems