Loss of Dnmt3a increases self-renewal and resistance to pegIFN-α in JAK2-V617F-positive myeloproliferative neoplasms.

ABSTRACT: Pegylated interferon alfa (pegIFN-α) can induce molecular remissions in patients with JAK2-V617F-positive myeloproliferative neoplasms (MPNs) by targeting long-term hematopoietic stem cells (LT-HSCs). Additional somatic mutations in genes regulating LT-HSC self-renewal, such as DNMT3A, have been reported to have poorer responses to pegIFN-α. We investigated whether DNMT3A loss leads to alterations in JAK2-V617F LT-HSC functions conferring resistance to pegIFN-α treatment in a mouse model of MPN and in hematopoietic progenitors from patients with MPN. Long-term treatment with pegIFN-α normalized blood parameters and reduced splenomegaly and JAK2-V617F chimerism in single-mutant JAK2-V617F (VF) mice. However, pegIFN-α in VF;Dnmt3aΔ/Δ (VF;DmΔ/Δ) mice worsened splenomegaly and failed to reduce JAK2-V617F chimerism. Furthermore, LT-HSCs from VF;DmΔ/Δ mice compared with VF were less prone to accumulate DNA damage and exit dormancy upon pegIFN-α treatment. RNA sequencing showed that IFN-α induced stronger upregulation of inflammatory pathways in LT-HSCs from VF;DmΔ/Δ than from VF mice, indicating that the resistance of VF;DmΔ/Δ LT-HSC was not due to failure in IFN-α signaling. Transplantations of bone marrow from pegIFN-α-treated VF;DmΔ/Δ mice gave rise to more aggressive disease in secondary and tertiary recipients. Liquid cultures of hematopoietic progenitors from patients with MPN with JAK2-V617F and DNMT3A mutation showed increased percentages of JAK2-V617F-positive colonies upon IFN-α exposure, whereas in patients with JAK2-V617F alone, the percentages of JAK2-V617F-positive colonies decreased or remained unchanged. PegIFN-α combined with 5-azacytidine only partially overcame resistance in VF;DmΔ/Δ mice. However, this combination strongly decreased the JAK2-mutant allele burden in mice carrying VF mutation only, showing potential to inflict substantial damage preferentially to the JAK2-mutant clone.

Iron is a modifier of the phenotypes of JAK2-mutant myeloproliferative neoplasms.

JAK 2-V617F mutation causes myeloproliferative neoplasms (MPNs) that can manifest as polycythemia vera (PV), essential thrombocythemia (ET), or primary myelofibrosis. At diagnosis, patients with PV already exhibited iron deficiency, whereas patients with ET had normal iron stores. We examined the influence of iron availability on MPN phenotype in mice expressing JAK2-V617F and in mice expressing JAK2 with an N542-E543del mutation in exon 12 (E12). At baseline, on a control diet, all JAK2-mutant mouse models with a PV-like phenotype displayed iron deficiency, although E12 mice maintained more iron for augmented erythropoiesis than JAK2-V617F mutant mice. In contrast, JAK2-V617F mutant mice with an ET-like phenotype had normal iron stores comparable with that of wild-type (WT) mice. On a low-iron diet, JAK2-mutant mice and WT controls increased platelet production at the expense of erythrocytes. Mice with a PV phenotype responded to parenteral iron injections by decreasing platelet counts and further increasing hemoglobin and hematocrit, whereas no changes were observed in WT controls. Alterations of iron availability primarily affected the premegakaryocyte-erythrocyte progenitors, which constitute the iron-responsive stage of hematopoiesis in JAK2-mutant mice. The orally administered ferroportin inhibitor vamifeport and the minihepcidin PR73 normalized hematocrit and hemoglobin levels in JAK2-V617F and E12 mutant mouse models of PV, suggesting that ferroportin inhibitors and minihepcidins could be used in the treatment for patients with PV.

JAK2-V617F and interferon-α induce megakaryocyte-biased stem cells characterized by decreased long-term functionality.

We studied a subset of hematopoietic stem cells (HSCs) that are defined by elevated expression of CD41 (CD41hi) and showed bias for differentiation toward megakaryocytes (Mks). Mouse models of myeloproliferative neoplasms (MPNs) expressing JAK2-V617F (VF) displayed increased frequencies and percentages of the CD41hi vs CD41lo HSCs compared with wild-type controls. An increase in CD41hi HSCs that correlated with JAK2-V617F mutant allele burden was also found in bone marrow from patients with MPN. CD41hi HSCs produced a higher number of Mk-colonies of HSCs in single-cell cultures in vitro, but showed reduced long-term reconstitution potential compared with CD41lo HSCs in competitive transplantations in vivo. RNA expression profiling showed an upregulated cell cycle, Myc, and oxidative phosphorylation gene signatures in CD41hi HSCs, whereas CD41lo HSCs showed higher gene expression of interferon and the JAK/STAT and TNFα/NFκB signaling pathways. Higher cell cycle activity and elevated levels of reactive oxygen species were confirmed in CD41hi HSCs by flow cytometry. Expression of Epcr, a marker for quiescent HSCs inversely correlated with expression of CD41 in mice, but did not show such reciprocal expression pattern in patients with MPN. Treatment with interferon-α further increased the frequency and percentage of CD41hi HSCs and reduced the number of JAK2-V617F+ HSCs in mice and patients with MPN. The shift toward the CD41hi subset of HSCs by interferon-α provides a possible mechanism of how interferon-α preferentially targets the JAK2 mutant clone.

HSP27 is a partner of JAK2-STAT5 and a potential therapeutic target in myelofibrosis.

Heat shock protein 27 (HSP27/HSPB1) is a stress-inducible chaperone that facilitates cancer development by its proliferative and anti-apoptotic functions. The OGX-427 antisense oligonucleotide against HSP27 has been reported to be beneficial against idiopathic pulmonary fibrosis. Here we show that OGX-427 is effective in two murine models of thrombopoietin- and JAKV617F-induced myelofibrosis. OGX-427 limits disease progression and is associated with a reduction in spleen weight, in megakaryocyte expansion and, for the JAKV617F model, in fibrosis. HSP27 regulates the proliferation of JAK2V617F-positive cells and interacts directly with JAK2/STAT5. We also show that its expression is increased in both CD34+ circulating progenitors and in the serum of patients with JAK2-dependent myeloproliferative neoplasms with fibrosis. Our data suggest that HSP27 plays a key role in the pathophysiology of myelofibrosis and represents a new potential therapeutic target for patients with myeloproliferative neoplasms.

Ruxolitinib-induced defects in DNA repair cause sensitivity to PARP inhibitors in myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPNs) often carry JAK2(V617F), MPL(W515L), or CALR(del52) mutations. Current treatment options for MPNs include cytoreduction by hydroxyurea and JAK1/2 inhibition by ruxolitinib, both of which are not curative. We show here that cell lines expressing JAK2(V617F), MPL(W515L), or CALR(del52) accumulated reactive oxygen species-induced DNA double-strand breaks (DSBs) and were modestly sensitive to poly-ADP-ribose polymerase (PARP) inhibitors olaparib and BMN673. At the same time, primary MPN cell samples from individual patients displayed a high degree of variability in sensitivity to these drugs. Ruxolitinib inhibited 2 major DSB repair mechanisms, BRCA-mediated homologous recombination and DNA-dependent protein kinase-mediated nonhomologous end-joining, and, when combined with olaparib, caused abundant accumulation of toxic DSBs resulting in enhanced elimination of MPN primary cells, including the disease-initiating cells from the majority of patients. Moreover, the combination of BMN673, ruxolitinib, and hydroxyurea was highly effective in vivo against JAK2(V617F)+ murine MPN-like disease and also against JAK2(V617F)+, CALR(del52)+, and MPL(W515L)+ primary MPN xenografts. In conclusion, we postulate that ruxolitinib-induced deficiencies in DSB repair pathways sensitized MPN cells to synthetic lethality triggered by PARP inhibitors.

Loss of Ezh2 synergizes with JAK2-V617F in initiating myeloproliferative neoplasms and promoting myelofibrosis.

Myeloproliferative neoplasm (MPN) patients frequently show co-occurrence of JAK2-V617F and mutations in epigenetic regulator genes, including EZH2 In this study, we show that JAK2-V617F and loss of Ezh2 in hematopoietic cells contribute synergistically to the development of MPN. The MPN phenotype induced by JAK2-V617F was accentuated in JAK2-V617F;Ezh2(-/-) mice, resulting in very high platelet and neutrophil counts, more advanced myelofibrosis, and reduced survival. These mice also displayed expansion of the stem cell and progenitor cell compartments and a shift of differentiation toward megakaryopoiesis at the expense of erythropoiesis. Single cell limiting dilution transplantation with bone marrow from JAK2-V617F;Ezh2(+/-) mice showed increased reconstitution and MPN disease initiation potential compared with JAK2-V617F alone. RNA sequencing in Ezh2-deficient hematopoietic stem cells (HSCs) and megakaryocytic erythroid progenitors identified highly up-regulated genes, including Lin28b and Hmga2, and chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) analysis of their promoters revealed decreased H3K27me3 deposition. Forced expression of Hmga2 resulted in increased chimerism and platelet counts in recipients of retrovirally transduced HSCs. JAK2-V617F-expressing mice treated with an Ezh2 inhibitor showed higher platelet counts than vehicle controls. Our data support the proposed tumor suppressor function of EZH2 in patients with MPN and call for caution when considering using Ezh2 inhibitors in MPN.

JAK2 exon 12 mutant mice display isolated erythrocytosis and changes in iron metabolism favoring increased erythropoiesis.

Mutations in JAK2 exon 12 are frequently found in patients with polycythemia vera (PV) that do not carry a JAK2-V617F mutation. The majority of these patients display isolated erythrocytosis. We generated a mouse model that expresses JAK2-N542-E543del, the most frequent JAK2 exon 12 mutation found in PV patients. Mice expressing the human JAK2-N542-E543del (Ex12) showed a strong increase in red blood cell parameters but normal neutrophil and platelet counts, and reduced overall survival. Erythropoiesis was increased in the bone marrow and spleen, with normal megakaryopoiesis and absence of myelofibrosis in histopathology. Erythroid progenitors and precursors were increased in hematopoietic tissues, but the numbers of megakaryocytic precursors were unchanged. Phosphorylation Stat3 and Erk1/2 proteins were increased, and a trend toward increased phospho-Stat5 and phospho-Stat1 was noted. However, Stat1 knock out in Ex12 mice induced no changes in platelet or red cell parameters, indicating that Stat1 does not play a central role in mediating the effects of Ex12 signaling on megakaryopoiesis or erythropoiesis. Ex12 mice showed decreased expression of hepcidin and increased expression of transferrin receptor-1 and erythroferrone, suggesting that the strong erythroid phenotype in Ex12 mutant mice is favored by changes in iron metabolism that optimize iron availability to allow maximal production of red cells.

Deletion of Stat3 in hematopoietic cells enhances thrombocytosis and shortens survival in a JAK2-V617F mouse model of MPN.

The acquired somatic JAK2-V617F mutation is present in >80% of patients with myeloproliferative neoplasms (MPNs). Stat3 plays a role in hematopoietic homeostasis and might influence the JAK2-V617F-driven MPN phenotype. We crossed our transgenic SclCre;V617F mice with a conditional Stat3 knockout strain and performed bone marrow transplantations into lethally irradiated recipient mice. The deletion of Stat3 increased the platelet numbers in SclCre;V617F;Stat3(fl/fl) mice compared with SclCre;V617F;Stat3(fl/+) or SclCre;V617F;Stat3(+/+) mice. Stat3 deletion also normalized JAK2-V617F-induced neutrophilia. Megakaryocyte progenitors were elevated, especially in the spleen, and a slight increase in myelofibrosis was noted. We observed increased mRNA expression levels of Stat1 and Stat1 target genes and augmented phosphorylation of Stat1 protein in bone marrow and spleen of JAK2-V617F mice after Stat3 deletion. The survival of Stat3-deficient mice expressing JAK2-V617F was reduced. Inflammatory bowel disease, previously associated with shortened survival of Stat3-deficient mice, was less prominent in the bone marrow transplantation setting, possibly by limiting deletion of Stat3 to hematopoietic tissues only. In conclusion, deletion of Stat3 in hematopoietic cells from JAK2-V617F mice did not ameliorate the course of MPN, but rather enhanced thrombocytosis and shortened the overall survival.

Haemorrhagic and thrombotic diatheses in mouse models with thrombocytosis.

We studied haemostasis in two mouse models with thrombocytosis caused by different pathogenic mechanisms. In one strain (Yall;Mpl-/-) thrombocytosis is driven by a misbalance between thrombopoietin and its receptor, whereas in the other strain, thrombocytosis is caused by expressing a human JAK2-V617F transgene (FF1) that depends on activation by Cre-recombinase (VavCre;FF1, MxCre;FF1). Thrombotic responses were increased following some, but not all types of challenges. In a vaso-occlusive thrombotic model following collagen-adrenaline injection we found increased mortality in both strains. Arterial thrombosis, examined after ferric chloride-induced carotid injury, was accelerated but with little impact on maximal thrombus size. In a vena cava stasis model, clots were of similar size as in wild-type controls, but exhibited a different composition with a higher platelet to fibrin ratio. Both thrombocytosis strains displayed increased haemorrhagic tendency in a tail bleeding assay. Yall;Mpl and VavCre;FF1 displayed a lower proportion of the more reactive high-molecular-weight forms of von Willebrand factor in their plasma, mimicking essential thrombocythaemia with very high platelet counts. Bleeding could not be explained by clear defects in platelet activation, which were normal or only weakly decreased. In conclusion, these models of thrombocytosis recapitulate several features of the haemorrhagic and thrombotic diatheses in ET and PV demonstrating potentials but also some limitations to study these major complications.

Estrogen signaling selectively induces apoptosis of hematopoietic progenitors and myeloid neoplasms without harming steady-state hematopoiesis.

Estrogens are potent regulators of mature hematopoietic cells; however, their effects on primitive and malignant hematopoietic cells remain unclear. Using genetic and pharmacological approaches, we observed differential expression and function of estrogen receptors (ERs) in hematopoietic stem cell (HSC) and progenitor subsets. ERα activation with the selective ER modulator (SERM) tamoxifen induced apoptosis in short-term HSCs and multipotent progenitors. In contrast, tamoxifen induced proliferation of quiescent long-term HSCs, altered the expression of self-renewal genes, and compromised hematopoietic reconstitution after myelotoxic stress, which was reversible. In mice, tamoxifen treatment blocked development of JAK2(V617F)-induced myeloproliferative neoplasm in vivo, induced apoptosis of human JAK2(V617F+) HSPCs in a xenograft model, and sensitized MLL-AF9(+) leukemias to chemotherapy. Apoptosis was selectively observed in mutant cells, and tamoxifen treatment only had a minor impact on steady-state hematopoiesis in disease-free animals. Together, these results uncover specific regulation of hematopoietic progenitors by estrogens and potential antileukemic properties of SERMs.

Myeloproliferative neoplasms can be initiated from a single hematopoietic stem cell expressing JAK2-V617F.

The majority of patients with myeloproliferative neoplasms (MPNs) carry a somatic JAK2-V617F mutation. Because additional mutations can precede JAK2-V617F, it is questioned whether JAK2-V617F alone can initiate MPN. Several mouse models have demonstrated that JAK2-V617F can cause MPN; however, in all these models disease was polyclonal. Conversely, cancer initiates at the single cell level, but attempts to recapitulate single-cell disease initiation in mice have thus far failed. We demonstrate by limiting dilution and single-cell transplantations that MPN disease, manifesting either as erythrocytosis or thrombocytosis, can be initiated clonally from a single cell carrying JAK2-V617F. However, only a subset of mice reconstituted from single hematopoietic stem cells (HSCs) displayed MPN phenotype. Expression of JAK2-V617F in HSCs promoted cell division and increased DNA damage. Higher JAK2-V617F expression correlated with a short-term HSC signature and increased myeloid bias in single-cell gene expression analyses. Lower JAK2-V617F expression in progenitor and stem cells was associated with the capacity to stably engraft in secondary recipients. Furthermore, long-term repopulating capacity was also present in a compartment with intermediate expression levels of lineage markers. Our studies demonstrate that MPN can be initiated from a single HSC and illustrate that JAK2-V617F has complex effects on HSC biology.

Loss of Stat1 decreases megakaryopoiesis and favors erythropoiesis in a JAK2-V617F-driven mouse model of MPNs.

The interferon-γ (IFNγ)/signal transducer and activator of transcription 1 (Stat1) pathway shows higher activity in patients with essential thrombocythemia (ET) than in polycythemia vera (PV) and was proposed to be promoting the ET phenotype. We explored the phenotypic consequences of Stat1 deficiency on the effects of Janus kinase 2 (JAK2)-V617F in vivo by crossing mice expressing JAK2-V617F with Stat1 knockout mice. JAK2-V617F;Stat1(-/-) double transgenic mice showed higher red cell parameters and lower platelet counts compared with JAK2-V617F;Stat1(+/+) mice. Bone marrow transplantation reproduced these phenotypic changes in wild-type recipients, demonstrating that the effect of Stat1 is cell-intrinsic and does not require a Stat1-deficient microenvironment. Deletion of Stat1 increased burst-forming unit-erythroid and reduced colony-forming unit-megakaryocyte colony formation driven by JAK2-V617F, but was not sufficient to completely normalize the platelet count. Gata1, a key regulator of megakaryopoiesis and erythropoiesis, was decreased in Stat1-deficient platelets. V617F transgenic mice with thrombocytosis had higher serum levels of IFNγ than normal controls and patients with ET showed higher IFNγ serum levels than patients with PV. Together, these results support the concept that activating Stat1 in the presence of JAK2-V617F, for example, through IFNγ, constrains erythroid differentiation and promotes megakaryocytic development, resulting in ET phenotype.

Differential effects of hydroxyurea and INC424 on mutant allele burden and myeloproliferative phenotype in a JAK2-V617F polycythemia vera mouse model.

To establish a preclinical animal model for testing drugs with potential effects on myeloproliferative neoplasms (MPNs), we first performed a detailed phenotypic characterization of Cre-inducible transgenic JAK2-V617F mice. Deleting the conditional mouse Jak2-knockout alleles increased erythropoiesis and accentuated the polycythemia vera phenotype, but did not alter platelet or granulocyte levels. In a transplantation assay, JAK2-V617F(+) BM cells had an advantage over wild-type competitor cells. Using this competitive repopulation assay, we compared the effects of INC424 (ruxolitinib), a dual Jak1/Jak2 inhibitor, and hydroxyurea (HU). HU led to weight loss, but did not reduce spleen weight. The hematologic parameters were lowered and a slight decrease of the mutant allele burden was noted. INC424 had little effect on body weight, but strongly decreased spleen size and rapidly normalized RBC and neutrophil parameters. No significant decrease in the mutant allele burden was observed. INC424 reduced the phospho-Stat5 levels, whereas HU strongly increased phospho-Stat5, most likely because of the elevated erythropoietin levels in response to the HU-induced anemia. This compensatory increase in JAK/STAT signaling may counteract the beneficial effects of cytoreduction at higher doses of HU and represents an adverse effect that should be avoided.

Regulation of gene expression of catecholamine biosynthetic enzymes in dopamine-beta-hydroxylase- and CRH-knockout mice exposed to stress.

Norepinephrine-deficient mice harbor a disruption of the gene for dopamine-beta-hydroxylase (DBH-KO). Corticotropin-releasing hormone knockout mice (CRH-KO) have markedly reduced HPA activity. The aim of the present work was to study how deficiency of DBH and CRH would affect tyrosine hydroxylase (TH), DBH, and phenylethanolamine N-methyltransferase (PNMT) gene expression and protein levels in the adrenal medulla (AM) and stellate ganglia (SG) of control and stressed mice. Both in AM and SG, single immobilization significantly increased TH and DBH mRNA and protein levels both in wild-type (WT) and CRH-KO mice. On the other hand, the stress-triggered increase in PNMT mRNA and protein levels seen in WT mice was absent in CRH-KO mice. DBH-KO mice are more sensitive to stress but survive a single 2 h restraint stress in a tube. The increase in TH mRNA levels induced by restraint stress in WT was not observed in DBH-KO mice. PNMT mRNA and especially PNMT protein levels were significantly elevated in AM of DBH-KO mice. In SG of DBH-KO mice, TH mRNA levels were not affected; however, PNMT gene expression was highly elevated. Thus, disruption of the DBH gene surprisingly blocks the stress-induced elevation of TH mRNA levels in AM but increases PNMT gene expression in both AM and SG. Our data indicate that adrenergic signaling is required for stress-induced increase in TH mRNA and that this signaling restrains stress-induced increase in PNMT mRNA. They also confirm that the HPA system plays a crucial role in the stress-induced regulation of PNMT gene expression.

Ethanol and stress activate catecholamine synthesis in the adrenal: effects on bone.

Ethanol consumption and mental stress activate the sympathetic nervous system, which can adversely affect bone. We compared six groups of 10 young adult rats, three with and three without 2 h daily restraint stress. Two groups consumed food and water ad libitum, two received food and 6% (w/v) ethanol as drinking water, and two received the amount of food consumed by ethanol rats the previous day plus water ad libitum (pairfed). After 6 weeks, rats were killed. Plasma, femurs, lumbar vertebrae, and adrenals were harvested. Femoral dimensions were measured and biomechanical properties were tested by three-point bending. Plasma osteocalcin, vertebral osteocalcin mRNA levels, and adrenomedullary tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and phenylethanolamine N-methyl transferase (PNMT) mRNA levels were quantified. Daily restraint decreased weight gain and femoral length compared to dietary controls, and appeared to partially preserve bone strength, especially in calorie-restricted pairfed rats. Femoral strength was significantly affected by treatment in that bones of pairfed controls were weakest, ethanol drinkers were intermediate, and ad libitum restrained were strongest. Femoral yield load, displacement, and work at yield load were negatively correlated with TH and DBH mRNA levels, but not PNMT, suggesting a negative influence of norepinephrine. Plasma osteocalcin and dry weight of lumbar 3-5 vertebrae were unaffected; however, osteocalcin mRNA in second lumbar vertebrae was positively correlated with TH, DBH, and PNMT levels. Ethanol consumption at this level had little effect on femur morphology or strength. In contrast, the data suggested possible stimulation rather than inhibition of vertebral bone formation.

Muscarinic receptors are affected by corticotropin-releasing hormone and c-fos gene disruptions: is there a mutual connection to adrenoceptors?

In the last decade, progress in gene disruption technology has allowed the study of the effects of the single-gene knockout (KO) on different molecules involved in the signaling cascade activated via muscarinic receptors. Many KO mice targeting muscarinic receptors have been developed, that is, all (M1-M5) muscarinic receptor KO mice (Wess, 2003) and acetylcholinesterase (AChE) KO mice(Xie et al., 2000). Recently, we have shown that these (AChE-/-) mice not only reveal changes in the number of muscarinic receptors in the heart, lung, cortex, and cerebellum but also in the number of adrenoceptors (Teplicky et al., 2004). Next, we studied whether the disruption of corticotropin-releasing hormone (CRH) or c-Fos could affect the properties of muscarinic receptors and adrenoceptors in the lungs and hearts of mice. The effects of immobilization stress in CRH KO animals were also studied.

Adrenergic modulation of the type 1 IP3 receptors in the rat heart.

Inositol 1,4,5-trisphosphate (IP3) receptors are calcium-releasing channels localized on the sarcoplasmic reticulum. IP3 receptors mediate the calcium mobilizing effect of a wide range of hormones, cytokines, and neurotransmitters and play an important role in variety of cell functions. The aim of this work was to study, how partial depletion of catecholamines affects the gene expression and protein levels of the type 1 IP3 receptors in rat heart. The type 1 IP3 receptor mRNA levels were studied in the left cardiac atrium and ventricle of rats treated with 6-hydroxydopamine (6-OHDA) in control and stressed conditions. The 6-OHDA produces anatomical and functional denervation resulting in decreased levels of noradrenaline and adrenaline. We also used corticoliberin (CRH) knockout mice, where secretion of adrenaline is significantly suppressed. Administration of 6-OHDA significantly decreases mRNA levels of the type 1 IP3 receptor in both, the left atrium and the left ventricle, while the gene expression of the sarcoplasmic reticular Ca2+-ATPase (SERCA 2) was unaffected. CRH knockout mice possess markedly lower levels of the type 1 IP3 receptor mRNA compared to wild-type mice in both, control and stressed conditions. These data point to the adrenergic modulation of the type 1 IP3 receptors in the rat hearts.

Ethanol consumption increases rat stress hormones and adrenomedullary gene expression.

Numerous reports document altered drinking behavior following acute stressors but few describe physiological responses to acute stress of chronic ethanol consuming subjects. We tested rats' responses to 120-min foot restraint immobilization (Immo) after 1 week of liquid diet containing 5% wt/vol ethanol (ethanol-fed). Controls consumed isocaloric liquid diet ad libitum (adlib-fed) or in amounts equal to that of ethanol-fed subjects on the previous day (pair-fed). Each rat was implanted with a tail artery cannula on day 7 to allow remote blood collection before and during Immo on day 8. Plasma epinephrine (Epi); norepinephrine (NE); corticosterone (Cort); prolactin (PRL); adrenomedullary gene expression of catecholamine biosynthetic enzymes tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and phenylethanolamine-N-methyl transferase (PNMT); and TH protein levels were measured. Ethanol-fed rats had two to threefold higher basal plasma Epi and NE and tended to have increased Cort compared to adlib-fed or pair-fed rats. Immo increased Epi and NE in ethanol-fed rats more than twofold above those observed in controls, and also increased Cort more in ethanol-fed than in control rats. PRL was marginally affected. Ethanol potentiated the normal immobilization-induced increase in adrenomedullary TH, DBH, and PNMT messenger RNA (mRNA). TH protein increased only in ethanol-fed rats. Increased plasma catecholamine levels, adrenomedullary gene expression, and TH protein concentration in nonimmobilized ethanol-fed rats strongly suggest that ethanol consumption was itself a stressor, which potentiated the subsequent response to acute Immo. Moreover, the observed interaction of ethanol and stress on plasma catecholamine levels illustrates the importance of minimizing additional stressful stimuli when investigating ethanol's physiological effects.

Social hierarchy affects gene expression for catecholamine biosynthetic enzymes in rat adrenal glands.

Social stressors, like other stressors, are powerful activators of the sympathoadrenomedullary system. Differential housing (single vs. group) and social defeat of rats is known to alter the activity of catecholamine-synthesizing enzymes in the medulla. The present studies examined the effect of 70 days of triad (3 rats per large cage) and individual housing of male rats on adrenal mRNA levels of tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT) and on TH protein levels. Behavioral ratings carried out at the triad formation indicated that dominant rats exhibited mostly offensive aggressive behaviors. By contrast, subordinate rats expressed primarily defensive behaviors, while the subdominant rats displayed intermediate levels of these behaviors. Overall, compared with single housing, triad housing resulted in lower gene expression for TH, DBH and PNMT and lower TH protein in the adrenals. Within triads, gene expression for these enzymes and TH protein concentration were higher in subordinate compared with dominant and subdominant rats. The dominant rats tended to have the lowest gene expression of these enzymes. These data indicate that in rodents, individual housing and a subject's social rank have a differential impact on the regulation of catecholamine biosynthesis already during the process of gene expression of catecholamine biosynthetic enzymes in the adrenals.

Immobilization stress-induced increase in plasma catecholamine levels is inhibited by a prolactoliberin (salsolinol) administration.

Catecholamines (CAs) are significantly involved in the regulation of homeostasis of the organism at rest and especially during stressful situations. Stress induces increases in plasma CA (epinephrine and norepinephrine) levels and prolactin (PRL) release from the adenopituitary. We have recently observed that salsolinol, which is produced by the neuro-intermediate lobe of the pituitary gland and by the hypothalamus, can selectively release PRL. Salsolinol is therefore considered to be a putative endogenous PRL-releasing factor. Based on the similarity of CA and PRL responses to stressors, we investigated whether salsolinol plays a role in the regulation of plasma CA levels at rest and of CA release induced by immobilization stress (IMO). Salsolinol did not affect CA baseline levels; however, it did inhibit IMO-induced CA release. Thus, the present study shows for the first time that salsolinol is not only a PRL-releasing factor but is also a potent inhibitor of stress-induced release of epinephrine and norepinephrine.