Studies on the prolactin-releasing mechanism of histones H2A and H2B

In previous studies we demonstrated that histone preparations possess multi ple effects in vivo on pituitary hormone secretion. We have now studied the specificity and signal transduction pathways involved in the prolactin (PR L)-releasing activity of histones H2A and H2B on perifused and incubated ra t pituitary cells. In the perifusion experiments, freshly dispersed pituita ry cells were packed into short columns and were continuously perifused wit h serum-free medium. The substances to be tested (stimuli) were pumped thro ugh the perifusion circuit, at the end of which perifusate fractions were c ollected and PRL measured by specific RIA. In the incubation studies, fresh ly dispersed pituitary cells were incubated in a metabolic incubator with d ifferent stimuli at different doses and for varying times. Perifusion of ce lls with median eminence extract (1/30), histone H2A (30 mu M) or histone H 2B (30 mu M), generated clear PRL release responses. Cells incubated with h istone H2A and H2B showed a dose- and time-dependent stimulatory effect on PRL release which, for H2A, was blocked by peptide MB-35, an 86-120 amino a cid synthetic fragment of histone H2A. The polycation, poly-lys was unable to mimic the action of histones. To detect the possible signal transduction pathways involved in the response of lactotrophs to histones, cells were i ncubated with the calcium ionophore A23187, the calcium chelator EGTA, the intracellular phosphoinositide enhancer LiCl, the intracellular cAMP enhanc ers caffeine, NaF and forskolin, and the protein kinase C inhibitor, triflu operazine (TFP). Both EGTA (or EGTA plus A23187 ionophore) and TFP were abl e to reduce significantly the response of lactotrophs to histones. Our resu lts confirm previous evidence that histones may act as hypophysotropic sign als. The data also suggest that calcium and diacylglycerol-associated pathw ays participate in these effects.