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H1 histones are involved in the formation of higher order chromatin structures and in the modulation of gene expression. Changes in chromatin structure are a characteristic initial feature of apoptosis. We therefore have investigated the histone H1 pattern of the human leukemic cell line HL60 undergoing programmed cell death, as induced by topoisomerase I inhibition. Histone H1 proteins were isolated and analyzed by high performance liquid chromatography and capillary zone electrophoresis. DNA fragmentation after apoptosis induction was monitored by agarose gel electrophoresis. The patterns of the three H1 histone subtypes extractable from apoptotic HL60 cells significantly differed from those of control cells in showing a decrease of phosphorylated H1 subtypes and an increase of the respective dephosphorylated forms. This dephosphorylation of H1 histones could be observed already 45 min after apoptosis induction and preceded internucleosomal DNA cleavage by approximately 2 h. We conclude that during apoptotic DNA fragmentation, the H1 histones become rapidly dephosphorylated by a yet unknown protein phosphatase.

Despite a rapidly increasing acceptance for a role of ATP as an extracellular mediator in several biological systems, the present report shows The role of nucleotides in metabolism is well established, but recognition of their potential importance as extracellular transmitters, regulators or modulators has been very recent. Extracellular ATP has been shown to induce a variety of responses, such as stimulation of inositol phospholipid turnover and activation of membrane conductances in many cell types and preparations (1-4). These responses are now known to be mediated by a family of ATP receptors, designated P2 purinoceptors (5-7), some of which have been cloned (8-11). ATP has been shown to stimulate inositol phosphate accumulation and intracellular Ca2+ metabolism in pituitary cell cultures (12), and our own data from fetal hypothalamic neuron cultures suggests that ATP has a possible regulatory role in the neuroendocrine system (13). Recently we have shown that ATP and UTP act on ATP receptors to provoke a rapid and dramatic increase in cytosolic Ca2+ in pituitary gonadotropin-release hormone (GnRH)-responsive cells (14). Using a superfusion system to examine the functional effect of extracellular nucleotides on the kinetics of pituitary hormone secretion and using real-time dynamic bioluminescence measurements of ATP, we now demonstrate that ATP receptors can mediate significant release of pituitary gonadotropin and that ATP can be exocytotically released from pituitary cells.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.MATERIALS AND METHODS Materials. Collagenase was purchased from Serva; medium M199, Ca2+-free Hanks' balanced salt solution, and sera were from GIBCO/BRL; GnRH and A23187 were from Calbiochem; and 2-methylthio-ATP and adenosine 5'-[3,y-methylene]triphosphate (13,y-methylene-ATP) were from Research Biochemical International (Natick, MA). Bromo-A23187, highly purified nucleotides, and all others, unless otherwise stated, were obtained from Sigma. Superfusion of Pituitary Cells. Pituitary glands from4-week-old female Wistar rats were enzymatically dispersed in medium M199 containing bovine serum albumin (BSA; 0.3%), collagenase (1 mg/ml), hyaluronidase (0.5 mg/ml), and DNase (25 ,ug/ml) as described (15). Aliquots of the cell suspension containing -3-4 x 106 cells were plated out on a 12-well culture plate containing 15 mg of Cytodex I beads, which had been preswollen in phosphate-buffered saline and autoclaved. Superfusion experiments were performed as described (15). After 2 days in culture, cells and beads were transferred to superfusion columns (5 x 25 mm Bio-Rad Econocolumns) and superfused with medium M199 containing 0.3% BSA, 1.8 mM Ca2+, 2 mM glutamine, and 20 mM Hepes at 37°C at the rate of 0.65 ml/min. A 90-min superfusion was carried out to wash cells before the start of superfusate collection. Superfusate fractio...

Natriuretic peptides act via receptors with intrinsic guanylate cyclase activity to stimulate cGMP production and are thought to be important regulators of neuroendocrine systems. C-Type natriuretic peptide (CNP) is of particular interest in this regard because the highest tissue concentrations of CNP occur in the anterior pituitary, where it is a highly potent stimulator of cGMP production. Here we show that pituitaries of rats and mice contain abundant CNP prohormone messenger RNA (mRNA), but no atrial natriuretic peptide or B-type natriuretic peptide prohormone mRNAs. Using reverse transcriptase-polymerase chain reaction, both A- and B-type natriuretic peptide receptor (GC-A and GC-B, respectively) transcripts were detected in rat and mouse pituitaries, although only the GC-B mRNA was measurable by Northern blotting. Immunohistochemistry revealed CNP-positive cells in the anterior, but not posterior, pituitaries of rats, and the vast majority of these cells were identified as gonadotropes by colocalization of CNP and LH immunoreactivities. Targeted toxicity using GnRH conjugated to the ricin-A chain was used to test whether gonadotropes are also direct targets for GnRH action. The conjugate dose dependently inhibited the proliferation of alpha T3-1 cells (gonadotrope-derived cells with GnRH receptors), but had no such effect on GH3 cells (which do not have GnRH receptors). Culture of rat pituitary cells with the conjugate caused comparable reductions in CNP-stimulated cGMP production, GnRH-stimulated LH release, and CA2+ ionophore (A23187)-stimulated LH release, but did not measurably alter cAMP production in response to pituitary adenylate cyclase-activating polypeptide. We conclude that CNP is synthesized in the pituitary, where it is located predominantly in gonadotropes, and GC-B receptors expressed in the pituitary mediate the direct effects of CNP in gonadotropes. Together with the recent demonstration of CNP synthesis and action in alpha T3-1 cells, the data suggest CNP to be a novel autocrine regulator of gonadotropes.