H1 and H2 histamine receptors mediate the production of inositol phosphates but not cAMP in human breast epithelial cells.

OBJECTIVE: In the present work we studied the H1 and H2 histamine receptor expression and function in HBL-100 and MCF-10A cells, derived from non-tumorigenic human breast epithelia, and in MCF-10T, the H-ras-transfected MCF-10A counterpart. The signal transduction pathways associated with these receptors, and the expression of proto-oncogenes c-fos, c-myc and c-jun at the mRNA and protein levels, were examined. RESULTS: Saturation analysis using intact cells, showed two binding sites for [3H]tiotidine and [3H]mepyramine. Pretreatment of purified membrane with guanosine 5'-ythiotriphosphate resulted in the loss of the low affinity component for [3H]tiotidine binding, and of the high affinity component for [3H]mepyramine. In both cases, there was no modification in the total number of sites for both ligands. Neither H1 nor H2 agonists stimulated cyclic AMP production, though this pathway is functional in these cells. On the other hand, both H1 and H2 agonists enhanced phosphoinositide turnover in a dose-dependent fashion, and this induction is pertussis toxin-insensitive. H1 and H2 agonists did not influence the expression of c-myc or c-fos mRNA, nor their encoded proteins. CONCLUSIONS: These results indicate that the three cell lines examined showed functional H1 and H2 histamine receptors, which are involved in the metabolic turnover of inositol phosphates but are ineffective in the modulation of the cyclic AMP response. The fact that H2 receptors have lost their ability to stimulate cyclic AMP production would imply the loss of a regulatory mechanism of cell growth.

Histamine H2 receptor desensitization: involvement of a select array of G protein-coupled receptor kinases.

The histamine H2 receptor (H2r) belongs to the heptahelical receptor family; upon agonist binding, members of this family activate a G protein and the downstream effector adenylyl cyclase. Like other G protein-coupled receptors, exposure of H2r to agonists produces a desensitization of the response. The present study focused on the desensitization mechanism of this receptor. Using transiently transfected COS-7 cells expressing tagged-H2r, the desensitization induced by amthamine, characterized by decreased cAMP production, was studied. Results show that the receptor was rapidly desensitized with a t(1/2) = 0.49 +/- 0.01 min. Because of the rapid nature of H2r desensitization, receptor phosphorylation was examined as a likely mechanism for signal attenuation. H2r desensitization was not affected by protein kinases A and C (PKA and PKC) inhibitors but was remarkably reduced by Zn(2+), an inhibitor of G protein-coupled receptor kinases (GRKs). Cotransfection experiments using tagged H2r and different GRKs (2, 3, 5, or 6), demonstrated that GRK2 and GRK3 were the most potent in augmenting desensitization, causing a reduction in the maximal response to amthamine and a decrease of the t(1/2) for desensitization, whereas GRK5 and GRK6 did not affect the signaling. Receptor phosphorylation correlates with desensitization for each GRK studied, whereas phosphorylation that is dependent on protein kinases A and C seemed irrelevant in receptor signal termination. These results indicate that in H2r-transfected COS-7 cells, exposure to an agonist caused desensitization controlled by H2r phosphorylation via GRK2 and GRK3.

Histamine modulates the expression of c-fos through cyclic AMP production via the H2 receptor in the human promonocytic cell line U937.

We examined the effects of histamine and its agonists on the expression of the c-fos and c-myc proto-oncogenes at the transcriptional and translational levels in the human promonocytic U937 cell line. Histamine transiently increased cAMP and c-fos expression through H2 receptors. Dibutyryl cAMP also increased c-fos mRNA and protein, and levels remained elevated even after 12 hr of treatment. Dose-dependence studies using histamine and dimaprit showed that the EC50 values for cAMP production and c-fos increase were similar, suggesting that cAMP might be involved in c-fos induction via H2 receptors. Furthermore, studies carried out using H7, a protein kinase A/protein kinase C inhibitor, blocked c-fos induction, whereas no effect was observed with bisindolylmaleimide, a specific protein kinase C inhibitor. No modification of c-myc expression could be detected on treatment with histamine or its analogues. Nevertheless, dibutyryl cAMP induced a down-regulation of the levels of this proto-oncogene. In addition, dibutyryl cAMP inhibited cell growth in a dose-dependent manner, whereas histamine failed to affect proliferation and differentiation of U937 cells. Cells pretreated with dimaprit showed a decrease in the cAMP response to subsequent addition of H2 agonists, whereas the cAMP response to prostaglandin E2 remained unaltered. This homologous mechanism of H2 receptor desensitization was time dependent. These results indicate that histamine activates several mechanisms involved in the induction of differentiation, such as cAMP and c-fos production, but fails to promote differentiation of U937 cells, apparently due to the rapid desensitization of H2 receptors.

Differentiating agents modulate topoisomerase I activity in U-937 promonocytic cells.

Phorbol 12-myristate 13-acetate (PMA), N,N'-hexamethylenebisacetamide (HMBA) and retinoic acid induce cell differentiation in U-937 promonocytic cells. This report examines the effects of these agents on DNA topoisomerase I activity. A decrease in enzyme activity could be detected as early as 30 min after treatment with all three differentiating compounds and lasted at least 48 h. No alteration in the levels of DNA topoisomerase I transcript or protein was observed during these treatments. The results might be explained by post-translational events that render DNA topoisomerase type I less active.

Increment of DNA topoisomerases in chemically and virally transformed cells.

The activities of topoisomerases I and II were assayed in subcellular extracts obtained from nontumorigenic BALB/c 3T3 A31 and normal rat kidney (NRK) cell lines and from the same cells transformed by benzo[a]pyrene (BP-A31), Moloney (M-MSV-A31) and Kirsten (K-A31) sarcoma viruses, and simian virus 40 (SV-NRK). The enzymatic activity of topoisomerase I was monitored by the relaxation of negatively supercoiled pBR322 DNA and by the formation of covalent complexes between 32P-labeled DNA and topoisomerase I. Topoisomerase II activity was determined by decatenation of kinetoplast DNA (k-DNA). It was found that nuclear and cytoplasmic type I topoisomerase specific activities were higher in every transformed cell line than in the normal counterparts. These differences cannot be attributed to an inhibitory factor present in A31 cells. When chromatin was treated at increasing ionic strengths, the 0.4 M NaCl extract showed the highest topoisomerase I specific activity. Moreover, in this fraction the transformed cells exhibited the most significant increment in the enzymatic activity as compared with nontransformed cultures. Spontaneously transformed A31 cells showed topoisomerase I activity similar to that of extracts of cells transformed by benzo[a]pyrene. Topoisomerase II specific activity was also increased in SV-NRK cells, as judged by the assay for decatenation of k-DNA to yield minicircle DNA.