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.

Prostaglandin E 2 receptors (EPR) belong to the family of G-protein-coupled receptors with 7 transmembrane domains. They form a family of four subtypes, which are linked to different G-proteins. EPiR are coupled to G q , EP 2 and EP 4 R to G s and EP 3 R to G;. Different C-terminal splice variants of the bovine EP 3 R are coupled to different G-proteins. A mouse EP 3 R whose C-terminal domain had been partially truncated no longer showed agonist-induced Gj -protein activation and was constitutively active. In order to test the hypothesis that the C-terminal domain confers coupling specificity of the receptors on the respective G-proteins, a cDNA for a hybrid rEP 3 hEP 4 R, containing the N-terminal main portion of the Gj-coupled rat EP 3 ßR including the 7th transmembrane domain and the intracellular C-terminal domain of the G s -coupled human EP 4 R, was generated by PCR. HEK293 cells transiently transfected with the chimeric rEP 3 hEP4R cDNA expressed a plasma membrane PGE 2 binding site with a slightly lower K d value for PGE 2 but an identical binding profile for receptorspecific ligands as cells transfected with the native rat EP 3 ßR. In HepG 2 cells stably transfected with the chimeric rEP 3 hEP 4 R cDNA PGE 2 did not increase cAMP formation characteristic of G s coupling but attenuated the forskolin-stimulated cAMP synthesis characteristic of Gj coupling. This effect was inhibited by pre-treatment of the cells with pertussis toxin. Thus, the hybrid receptor behaved both in binding and in functional coupling characteristics as the native rat EP 3ß R. Apparently, the intracellular C-terminal domain did not confer coupling specificity but coupling control, i.e. allowed a signalling state of the receptor only with agonist binding.

H1 histone subtype genes differ in their expression patterns during the different stages of the cell cycle interphase. While the group of replication-dependent H1 histone subtypes is synthesized during S phase, the replacement histone subtype H1.0 is also expressed replication-independently in non-proliferating cells. The present study is the first report about the analysis of the cell cycle-dependent expression of all five replication-dependent H1 subtypes, the replacement histone H1.0 and the ubiquitously expressed subtype H1x. The expression of these H1 histone subtypes in HeLa cells was analyzed on mRNA level by quantitative real-time RT-PCR as well as on protein level by immunoblotting. We found that after arrest of HeLa cells in G(1) phase by treatment with sodium butyrate, the mRNA levels of all replication-dependently expressed H1 subtypes decreased, but to very different extent. During S phase the individual replication-dependently expressed H1 subtypes show similar kinetics regarding their mRNA levels. However, the variations in their protein amounts partially differ from the respective RNA levels which especially applies to histone H1.3. In contrast, the mRNA as well as the protein level of H1x remained nearly unchanged in G(1) as well as during S phase progression. The results of the present study demonstrate that the cell cycle-dependent mRNA and protein expression of various H1 subtypes is differentially regulated, supporting the hypothesis of a functional heterogeneity.

Rat hepatocytes have previously been reported to possess prostaglandin E, receptors of the EP,-type (EP,-receptors) that inhibit glucagonstimulated glycogenolysis by decreasing CAMP. Here, the isolation of a functional EP, receptor cDNA clone from a rat hepatocyte cDNA library is reported. This clone can be translated into a 362-amino-acid protein, that displays over 95% homology to the EP, receptor from mouse mastocytoma. The amino-and carboxy-terminal region of the protein are least conserved. Transiently transfected HEK 293 cells expressed a single binding site for PGE, with an apparent Kd of 15 nM. PGE, > PGF,, > PGDz competed for ['HIPGEl binding sites as did the EP, receptor agonists M&B 28767 = sulprostone > misoprostol but not the EP, receptor antagonist SC 19220. In stably transfected CHO cells M&B 28767 > sulprostone = PGE, > misoprostol > PGF, inhibited the forskolin-elicited CAMP formation. Thus, the characteristics of the EP, receptor of rat hepatocytes closely resemble those of the EP, receptor of mouse mastocytoma.

Rat liver Ito cells were cultured for 24 hr with 20% newborn calf serum. Stimulation with the sympathetic neurotransmitter noradrenaline (0.1 pmol/L to 1 mmoln) led to a dose-dependent increase in prostaglandin F,, release and a slightly smaller enhancement of prostaglandin D, production. Prostaglandin F,, and prostaglandin D, synthesis was highest in the first 30 sec after stimulation. Stimulation with the possible cotransmitter A W (10 pmol/L and 1 mmoln ATP) also enhanced both prostaglandin F,, and prostaglandin D, release strongly. The release was highest again during the first 30 sec. Stimulation with noradrenaline and ATP simultaneously did not increase the effects of noradrenaline or ATP alone. Adenosine had no effect on prostaglandin production. The effects of noradrendine were inhibited specifically by the a,-adrenoreceptor antagonist prazosin but not by the p,-purinoreceptor antagonist 8-phenyltheophylline. The effects of ATP were not antagonized by the inhibitors. Because the metabolic actions of sympathetic hepatic nerves can be inhibited by inhibitors of prostanoid synthesis and mimicked by prostaglandins F,, and D,, and because the Ito cells are well innervated, our results permit the conclusion that Ito cells could be involved in the nervous signal chain: During sympathetic nerve action the neurotransmitter noradrenaline and the cotransmitter ATP cause increases in prostaglandin The liver is innervated by sympathetic and parasympathetic, afferent and efferent nerves (1-5). Stimulation of the perivascular nerve bundles in isolated perfused rat liver led to increases in glucose and lactate output (6,7),

Posttranslational modifications of histones have an integral function in the structural and functional organization of chromatin. Several changes in the modification state of histones could be observed after induction of apoptosis with topoisomerase inhibitors and other inducers. Most of these studies include the analysis of the state of phosphorylation of histones, and the results are to some extent controversial, depending on cell lines and agents used. In the present study we compared the kinetics of the dephosphorylation of H1 and H3 histones with apoptosis markers after treatment of leukemic cell lines with topoisomerase inhibitors. In parallel, we determined cell cycle parameters in detail. Dephosphorylation of both histone classes started within 1 h of induction, and no direct correlation with timing and intensity of the investigated apoptotic features could be observed. In contrast, we show that the effect of topoisomerase inhibitors on the state of H1 and H3 phosphorylation is not directly related to apoptosis, but reflects the changes in the cell cycle distribution of cells treated with these inducers.

Prostaglandin E 2 receptors (EPR), which belong to the family of heterotrimeric G protein-coupled ectoreceptors with seven transmembrane domains, can be classified into four subtypes according to their ligand binding and G protein coupling specificity. Of these, EP 3 ßR is coupled to G¡, whereas EP 4 R is coupled to G s . EP 4 R, in contrast to EP 3 ßR, shows agonist-induced desensitization. The C-terminal domain and the third intracellular loop of these receptors have been implicated in G protein coupling specificity and desensitization. Here, receptor hybrids consisting of the main portion of rat EP 3 ßR and either the C-terminal domain or the third intracellular loop of human EP 4 R were used to study the contribution of the respective receptor domains to G protein coupling and desensitization. Neither the EP 4 R C-terminal domain nor the EP 4 R third intracellular loop alone was sufficient to change the coupling specificity of the rEP 3 hEP 4 receptor hybrids from G¡ to G s or to confer additional G s coupling. However, the EP 4 R C-terminal domain but not the third intracellular loop was necessary and sufficient to mediate rapid agonist-induced, second messengerindependent desensitization in the G¡-coupled hybrid receptors.