kn-62 and Astrocytoma

We examined the regulatory mechanisms of the affinity of Gq protein-coupled histamine H(1)-receptors for histamine after histamine pretreatment in intact human U373 MG astrocytoma cells. In control cells, the displacement curves for histamine against the binding of 5 nM [(3)H]mepyramine, a radioligand for H(1)-receptors, showed the presence of two binding sites for histamine, that is, high and low affinity sites. Pretreatment with 0.1 mM histamine for 30 min at 37°C induced a significant reduction in the percentage of high affinity sites for histamine and a concomitant increase in the percentage of low affinity sites with no change in their pIC(50) values. These histamine-induced changes were insensitive to 30 µM KN-62, an inhibitor of Ca(2+)/calmodulin-dependent protein kinase II, but they were completely inhibited either by 0.4 mM ZnCl(2), an inhibitor of G protein-coupled receptor kinases (GRKs), or under hypertonic conditions, where clathrin-mediated endocytosis is known to be inhibited. These results suggest that histamine-induced conversion of high to low affinity sites for histamine is predominantly regulated by GRK/clathrin-mediated internalization of H(1)-receptors in human astrocytoma cells.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Astrocytoma; Binding Sites; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Chlorides; Clathrin; Endocytosis; G-Protein-Coupled Receptor Kinases; GTP-Binding Protein alpha Subunits, Gq-G11; Histamine; Histamine H1 Antagonists; Humans; Receptors, Histamine H1; Tumor Cells, Cultured; Zinc Compounds

We investigated Ca(2+)/calmodulin (CaM)-mediated regulation of the desensitizing process of the histamine H(1) receptor-mediated increase in intracellular Ca(2+) concentration in human U373 MG astrocytoma cells. The desensitizing process was evaluated by measuring the histamine-induced Ca(2+) responses in cells pretreated with histamine for 15 s-30 min under various conditions. Under normal physiological conditions, desensitization developed with three successive phases : a fast desensitization within 15 s, a transient resensitization at 45 s, and a prompt and sustained redesensitization from 1 to 30 min. Similar processes of desensitization/resensitization occurred even under hypertonic conditions, where histamine-mediated internalization of the histamine H(1) receptor is inhibited. The transient resensitization phase was selectively prevented by deprivation of extracellular Ca(2+) and, even more strikingly, by the presence of W-7 (a CaM antagonist). FK506 and cyclosporin A, Ca(2+)/CaM-dependent protein phosphatase (PP2B) inhibitors, mimicked such effects. In the presence of KN-62, a Ca(2+)/CaM-dependent protein kinase II (CaM kinase II) inhibitor, the early development of desensitization disappeared, allowing a slow and simple development of desensitization. The early processes of desensitization and resensitization were unaffected by W-5, okadaic acid, and KN-04 (less potent inhibitors against CaM, PP2B, and CaM kinase II, respectively) or by GF109203X and chelerythrine (protein kinase C inhibitors). The high-affinity site for histamine was converted to a lower-affinity site by histamine treatment, which also showed a transient restoration phase at 45 s in a manner sensitive to KN-62 and FK506. These results provide the first evidence that Ca(2+)/CaM plays a crucial role in determining the early phase of the desensitizing process via activation of CaM kinase II and PP2B, by regulating agonist affinity for histamine H(1) receptors.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Astrocytoma; Calcineurin Inhibitors; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Cyclosporine; Enzyme Inhibitors; GTP-Binding Protein alpha Subunits, Gq-G11; GTP-Binding Proteins; Histamine; Histamine H1 Antagonists; Humans; Kinetics; Okadaic Acid; Piperidines; Pyrilamine; Ranitidine; Receptors, Histamine H1; Sulfonamides; Tacrolimus; Tumor Cells, Cultured