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The localization of condensin along chromosomes is crucial for their accurate segregation in anaphase. Condensin is enriched at telomeres but how and for what purpose had remained elusive. Here, we show that fission yeast condensin accumulates at telomere repeats through the balancing acts of Taz1, a core component of the shelterin complex that ensures telomeric functions, and Mit1, a nucleosome remodeler associated with shelterin. We further show that condensin takes part in sister-telomere separation in anaphase, and that this event can be uncoupled from the prior separation of chromosome arms, implying a telomere-specific separation mechanism. Consistent with a cis-acting process, increasing or decreasing condensin occupancy specifically at telomeres modifies accordingly the efficiency of their separation in anaphase. Genetic evidence suggests that condensin promotes sister-telomere separation by counteracting cohesin. Thus, our results reveal a shelterin-based mechanism that enriches condensin at telomeres to drive in cis their separation during mitosis., Competing Interests: LC, CR, JB, LM, LM, ET, NC, SS, OC, YG, SC, PB, ST No competing interests declared, (© 2023, Colin, Reyes et al.)

Background and Purpose: Phenelzine is an antidepressant drug known to increase the risk of hypertensive crisis when dietary tyramine is not restricted. However, this MAO inhibitor inhibits other enzymes not limited to the nervous system. Here we investigated if its antiadipogenic and antilipogenic effects in cultured adipocytes could contribute to decreased body fat in vivo, without unwanted hypertensive or cardiovascular effects., Experimental Approach: Mice were fed a standard chow and given 0.028% phenelzine in drinking water for 12 weeks. Body composition was determined by NMR. Cardiovascular dysfunction was assessed by heart rate variability analyses and by evaluation of cardiac oxidative stress markers. MAO activity, hydrogen peroxide release and triacylglycerol turnover were assayed in white adipose tissue (WAT), alongside determination of glucose and lipid circulating levels., Key Results: Phenelzine-treated mice exhibited lower body fat content, subcutaneous WAT mass and lipid content in skeletal muscles than control, without decreased body weight gain or food consumption. A modest alteration of cardiac sympathovagal balance occurred without depressed aconitase activity. In WAT, phenelzine impaired the lipogenic but not the antilipolytic actions of insulin, MAO activity and hydrogen peroxide release. Phenelzine treatment lowered non-fasting blood glucose and phosphoenolpyruvate carboxykinase expression. In vitro, high doses of phenelzine decreased both lipolytic and lipogenic responses in mouse adipocytes., Conclusion and Implications: As phenelzine reduced body fat content without affecting cardiovascular function in mice, it may be of benefit in the treatment of obesity-associated complications, with the precautions of use recommended for antidepressant therapy., (© 2018 The British Pharmacological Society.)

Tyramine is naturally occurring in food and induces pressor responses. Low-tyramine diets are recommended for patients treated with MAO inhibitors to avoid the fatal hypertensive crisis sadly known as "cheese effect". Hence, tyramine intake is suspected to have toxicological consequences in humans, while its administration to type 1 diabetic rodents has been reported to improve glucose tolerance. We investigated in mice whether prolonged tyramine ingestion could alter glucose homeostasis, insulin sensitivity, adipose tissue physiology or cardiovascular functions. Tyramine was added at 0.04 or 0.14 % in the drinking water since this was estimated to increase by 10- to 40-fold the spontaneous tyramine intake of control mice fed a standard diet. Ten to 12 weeks of such tyramine supplementation did not influence body weight gain, adiposity or food consumption. Both doses (reaching approx. 300 and 1100 μmol tyramine/kg bw/day) decreased nonfasting blood glucose but did not modify glucose tolerance or fasting levels of glucose, insulin or circulating lipids. Blood pressure was not increased in tyramine-drinking mice, while only the higher tested dose moderately increased heart rate without change in its variability. Markers of cardiac tissue injury or oxidative stress remained unaltered, except an increased hydrogen peroxide production in heart preparations. In isolated adipocytes, tyramine inhibited lipolysis similarly in treated and control groups, as did insulin. The lack of serious adverse cardiovascular effects of prolonged tyramine supplementation in normoglycemic mice together with the somewhat insulin-like effects found on adipose cells should lead to reconsider favourably the risk/benefit ratio of the intake of this dietary amine.

The objectives of this study were to determine whether type 2 diabetic mice would exhibit a more severe renal impact of hemorrhagic shock (HS) based on a recently described model of acute kidney injury and to determine the impact of HS on renal responses to hypoxia. We induced HS or sham procedure in type 2 diabetic and obese db/db mice. Creatininemia, glomerular filtration rate, urine output, histologic injury score, and kidney inductible molecule 1 mRNA were used to investigate the renal impact of HS. Tissular hypoxia and its impact were quantified using pimonidazole immunostaining and mRNA of hypoxic inducible factor, vascular endothelial growth factor receptors 1 and 2, Tie-2, endothelial nitric oxide synthase, and inducible nitric oxide synthase. Diabetic mice exhibiting mild diabetic nephropathy express hypoxic signals at baseline. The renal impact of HS was more severe in diabetic mice, with a worsening of tissular hypoxia and an altered response to hypoxia. Furthermore, endothelial nitric oxide synthase was highly overexpressed in diabetic shocked mice when compared with nondiabetic shocked mice. Renal impact of HS in type 2 diabetic mice is more intense than in nondiabetic ones. Preexisting hypoxia during diabetes could result in a renal preconditioning that modifies endothelial and tissular responses to acute kidney injury.

Aims: Activation of cardiac fibroblasts and their differentiation into myofibroblasts is a key event in the progression of cardiac fibrosis that leads to end-stage heart failure. Apelin, an adipocyte-derived factor, exhibits a number of cardioprotective properties; however, whether apelin is involved in cardiac fibroblast activation and myofibroblast formation remains unknown. The aim of this study was to determine the effects of apelin in activated cardiac fibroblasts, the potential related mechanisms and impact on cardiac fibrotic remodelling process., Methods and Results: In vitro experiments were performed in mouse cardiac fibroblasts obtained from normal and pressure-overload hearts. Pretreatment of naive cardiac fibroblasts with apelin (1-100 nM) inhibited Transforming growth factor-β (TGF-β)-mediated expression of the myofibroblast marker α-smooth muscle actin (α-SMA) and collagen production. Furthermore, apelin decreased the spontaneous collagen production in cardiac fibroblasts isolated from hearts after aortic banding. Knockdown strategy and pharmacological inhibition revealed that prevention of collagen accumulation by apelin was mediated by a reduction in sphingosine kinase 1 (SphK1) activity. In vivo studies using the aortic banding model indicated that pretreatment with apelin attenuated the development of myocardial fibrotic remodelling and inhibited cardiac SphK1 activity and α-SMA expression. Moreover, administration of apelin 2 weeks after aortic banding prevented cardiac remodelling by inhibiting myocyte hypertrophy, cardiac fibrosis, and ventricular dysfunction., Conclusion: Our data provide the first evidence that apelin inhibits TGF-β-stimulated activation of cardiac fibroblasts through a SphK1-dependent mechanism. We also demonstrated that the administration of apelin during the phase of reactive fibrosis prevents structural remodelling of the myocardium and ventricular dysfunction. These findings may have important implications for designing future therapies for myocardial performance during fibrotic remodelling, affecting the clinical management of patients with progressive heart failure.

Objective: Sparse data are available on renal consequences of hemorrhagic shock in mice. This study aimed to extend the current knowledge on functional and morphologic renal impact of hemorrhagic shock in mice and to determine its ability to stand as an accurate model of acute kidney injury., Design: In vivo study., Setting: University research unit., Subjects: C57/Bl6 mice., Interventions: A model of controlled hemorrhagic shock was adapted to determine the renal impact of hemorrhagic shock in mice., Measurements and Main Results: Renal functions and kidney morphology were followed up from 3 hrs to 21 days after hemorrhagic shock. When prolonged up to 2 hrs, hypotension (35 mm Hg mean arterial blood pressure) induced by temporary blood removal was responsible for an early and lasting increase in hypoxia-inducible factor-1α and kidney-inducible molecule-1 gene expression that paralleled acute tubular necrosis and renal failure. Two-hr hypotension induced an important but reversible decrease in glomerular filtration rate up to 6 days after hemorrhagic shock. Other renal dysfunctions included a renal loss of sodium, assessed by the increase in sodium excretion, and a decrease in urine concentration that persists up to day 21. Tissular damages prevailed in the outer medulla 2 days after hemorrhagic shock, being maximal at day 6. At day 21, renal healing was associated with epithelial recovery and a significant interstitial fibrosis., Conclusions: Our data indicate that apparent recovery of renal function after acute kidney injury can mask persisting dysfunctions and tissular damages that could predispose to chronic kidney disease. Prolonged hemorrhagic shock in mice closely mimics renal effects induced by similar situation in humans, thus providing a useful tool to investigate pathophysiological mechanisms and protection strategies against acute kidney injury in situations such as hemorrhagic shock.

The alpha(2B)-adrenoceptor (alpha(2B)-AR) mediates vasoconstriction and a common polymorphism (+901 Ins/Del), located in the coding region of the human alpha(2B)-AR gene (ADRA2B), has been demonstrated to affect receptor function in vitro. In this study, we have identified a novel polymorphism corresponding to the insertion of 12-nucleotides (GGGACGGCCCTG) at position -4825 relative to the start codon (-4825 del/ins) in the far upstream region of the ADRA2B promoter. The genotyping of 71 unrelated Finnish individuals showed that the -4825 ins polymorphism is common and in complete linkage with the Del polymorphism at position +901 and a G/C substitution at position -98. Transfection of various cell lines with luciferase constructs containing a 5.5 kb fragment of the ADRA2B promoter region indicated that the 12-nucleotide insertion at -4825 resulted in a large reduction of transcriptional activity. Electrophoretic mobility shift assays with oligonucleotide probes corresponding to the two ADRA2B alleles demonstrated that the region where the -4825 del/ins variation occurs binds nuclear proteins and that the 12-nucleotide insertion affects the pattern of bound transcription factors. Altogether, the present findings show that the previously identified +901 Del polymorphism is linked with a variation in the ADRA2B promoter that affects transcriptional activity in vitro. The molecular mechanisms underlying this effect are still unclear but a possible impact of the -4825 ins polymorphism on alpha(2B)-AR expression would merit to be examined in vivo as a diminution of promoter activity may limit the functional consequences of the +901 Del polymorphism.

alpha(2A)-adrenoceptors are expressed on intestinal cells and they participate in the control of epithelial functions such as solute and water transport or cell proliferation. In pathological conditions, pro-inflammatory cytokines secreted by lymphocytes are responsible for modification of intestinal cell characteristics including phenotype switch and changes in the expression of pumps and ion channels. Using the HT29 cell line as a model, the present work examined the effect of two inflammatory cytokines, interferon-gamma (IFNgamma) and tumor necrosis factor-alpha (TNFalpha), on the expression of the human alpha(2A)-adrenoceptor. Exposure of cells to either IFNgamma or TNFalpha resulted in a concentration- and time-dependent diminution of [(3)H]RX821002 binding sites, which is preceded by a large decrease in the amount of alpha(2A)-adrenoceptor mRNA. The cytokines did not affect the receptor mRNA half-life, but inhibited the activity of a luciferase construct containing the promoter region of alpha(2A)-adrenoceptor gene, indicating that a decrease in the transcription rate is primarily responsible for the diminution of receptor expression. Exposure of cells to either IFNgamma or TNFalpha caused increased production of reactive oxygen species and transient phosphorylation of extracellular signal-regulated kinase (Erk1/2). The effect of cytokines was mimicked by H(2)O(2) but was unaffected by the addition of anti-oxidants. The blockade of Erk1/2 activation by PD98059 blunted the effect of TNFalpha but not of IFNgamma. In conclusion, the present findings demonstrate that IFNgamma and TNFalpha diminish the alpha(2A)-adrenoceptor expression in HT29 cells by decreasing the transcription rate without modifying the stability of mRNA. The transcription inhibition is however triggered via different signalling pathways. The results suggest that cytokine-mediated down-regulation of alpha(2A)-adrenoceptor could contribute to the pathogenesis of inflammatory bowel disease.

Intestinal cells express alpha(2A)-adrenoreceptors that stimulate sodium and peptide absorption and promote cell proliferation. Involved mechanisms are poorly understood and are not fully related to inhibition of cAMP production. Previous study using a clone of CaCo2 cells expressing the human alpha(2A)-adrenoreceptor (CaCo2-3B) showed that alpha(2)-adrenoreceptor agonists cause extracellular signal-regulated kinase (ERK) phosphorylation. Present work examines the signaling pathway triggering ERK activation and investigates the consequence of alpha(2A)-adrenoreceptor stimulation on cell migration. Treatment of CaCo2-3B with the alpha(2)-adrenoreceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino) quinoxaline (UK14304) induces not only ERK, but also Akt phosphorylation. Both effects are strongly attenuated by inhibition or desensitization of epidermal growth factor (EGF) receptor, matrix metalloproteinase (MMP) blockade, heparin-binding-EGF neutralization or phosphatidylinositol 3-kinase (PI3-kinase) inhibitors. Conditioned medium from UK14304-treated CaCo2-3B stimulates ERK in parental CaCo2 by a mechanism sensitive to EGF receptor and PI3-kinase inhibitors. Exposure of CaCo2-3B to UK14304 accelerates the wound healing. This effect is abolished by heparin-binding-EGF neutralization but not by mitomycin C, indicating that it results probably from increased cell spreading and/or migration. In conclusion, alpha(2A)-adrenoreceptor activates ERK and Akt in intestinal cells by a common pathway which depends on PI3-kinase activation and results from EGF receptor transactivation, via an autocrine/paracrine pathway implying MMP activation and heparin-binding-EGF shedding. Therefore, alpha(2A)-adrenoreceptor could have a positive role in intestinal regeneration in vivo.

To assess the stereochemical requirements for efficient alpha2C-adrenoreceptor activation, the enantiomeric forms of m-nitrobiphenyline [(+/-)-5] were prepared and tested on cells expressing the human alpha2-adrenoreceptor subtypes. The importance of chirality was confirmed, since the enantiomer (R)-(+)-5 was much more efficient than (S)-(-)-5 in producing alpha2C-activation. Surprising reversal of enantioselectivity was observed with respect to structurally similar biphenyline [(+/-)-1] whose (S)-(-)-form proved the preferred alpha2C-configuration.

Adrenergic receptors (ARs) are directly or indirectly involved in the control of a large panel of physiological functions and are the targets of drugs for the treatment of several common diseases including congestive heart failure, asthma or benign prostatic hyperplasia. The genotyping of human populations with diverse ethnicity has revealed that the genes encoding alpha(1A)-, alpha(1B)-, alpha(2A)-, alpha(2B)-, alpha(2C)-, beta(1)-, beta(2)- and beta(3)-AR are polymorphic in their coding region as well as in their regulatory domains and non-coding regions. The functional consequences of these genetic variations include changes in expression at transcriptional or translational level, modification of coupling to heterotrimeric G-proteins resulting in a gain or a loss in function, and alteration of GRK-mediated receptor phosphorylation/desensitization or of agonist-promoted down-regulation. None of the mutations identified so far is per se a major risk factor for acquired or inherited disease; however, variants of alpha(2C)-AR and beta(1)-AR may act in synergy to determine the progression of heart failure and certain combinations of polymorphisms on beta(2)-AR correlate with asthmatic phenotypes or response to beta(2)-agonist therapy. Herein we summarize the present knowledge on AR gene polymorphisms, and discuss the putative consequences of variations resulting in receptor malfunction on pharmacogenomics and disease predisposition.

Activation of heterotrimeric G proteins by their cognate seven transmembrane domain receptors is believed to involve conformational changes propagated from the receptor to the G proteins. However, the nature of these changes remains unknown. We monitored the conformational rearrangements at the interfaces between receptors and G proteins and between G protein subunits by measuring bioluminescence resonance energy transfer between probes inserted at multiple sites in receptor-G protein complexes. Using the data obtained for the alpha(2A)AR-G alpha(i1) beta1gamma2 complex and the available crystal structures of G alpha(i1) beta1gamma2, we propose a model wherein agonist binding induces conformational reorganization of a preexisting receptor-G protein complex, leading the G alpha-G betagamma interface to open but not dissociate. This conformational change may represent the movement required to allow nucleotide exit from the G alpha subunit, thus reflecting the initial activation event.

Dietary cholesterol absorption contributes to a large part of the circulating cholesterol. However, the mechanism of sterol intestinal uptake is not clearly elucidated. Scavenger receptor class B type I (SR-BI), major component in the control of cholesterol homeostasis, is expressed in the intestine, but its role in this organ remains unclear. We have generated transgenic mice overexpressing SR-BI primarily in the intestine by using the mouse SR-BI gene under the control of intestinal specific "apoC-III enhancer coupled with apoA-IV promoter." We found SR-BI overexpression with respect to the natural protein along the intestine and at the top of the villosities. After a meal containing [(14)C]cholesterol and [(3)H]triolein, SR-BI transgenic mice presented a rise in intestinal absorption of both lipids that was not due to a defect in chylomicron clearance nor to a change in the bile flow or the bile acid content. Nevertheless, SR-BI transgenic mice showed a decrease of total cholesterol but an increase of triglyceride content in plasma without any change in the high density lipoprotein apoA-I level. Thus, we described for the first time a functional role in vivo for SR-BI in cholesterol but also in triglyceride intestinal absorption.

In the rat, the alpha2B-adrenergic receptor (alpha2B-AR) is encoded by the rat non-glycosylated (RNG) gene and is primarily expressed in the kidney, brain and liver of adult animals. High levels of alpha2B-AR are also found during fetal life in the placenta, liver and blood, where it is borne by cells of the erythropoietic lineage. As a first step to define the mechanisms responsible for the spatio-temporal pattern of alpha2B-AR expression, a genomic fragment containing 2.8 kb of the 5'-flanking region, the ORF and approximately 20 kb of the 3'-flanking region of the RNG gene was isolated. RNase protection assays performed on RNA from placenta or kidney using a series of riboprobes permitted to locate the transcription start site 372 bases upstream from the start codon. Transient transfection of various cells, including rat proximal tubule in primary culture, with constructs containing luciferase as a reporter gene demonstrated that: (i) the 5'-flanking region exhibited a strong and sense-dependent transcriptional activity and (ii) the 332 bp fragment (-732/-401 relative to the start codon), which lacks a TATA box but contains Sp1 sites, is sufficient to drive expression. Analysis of chromatin susceptibility to DNaseI digestion identified two hypersensitive sites (HS1 and HS2) located 1.7 and 1.0 kb, respectively, upstream from ATG and containing recognition sequences for erythroid transcription factors. EMSA showed specific binding of GATA1 and NF-E2 to these elements. Taken together, the results suggest that the chromatin environment in the vicinity of these boxes plays a critical role for alpha2B-AR expression during fetal life.

Screening of a foetal brain genomic DNA library allowed to isolate a 10-kb fragment of the gene encoding the human alpha2B-adrenergic receptor, that contained 5.5 kb of the 5'-flanking region, the open reading frame and 2.9 kb of the 3'-flanking region. The 1-kb fragment upstream from the start codon was rich in GC, lacked consensus TATA or CAAT box, but contained several Sp1-binding sites. Other potential cis-regulatory elements found in the 5'-flanking region included AP2, USF, Stat-6, NFkappaB and Olf-1. A single canonical polyadenylation signal (AATAAA) was found at position +3252/+3257 and the polyadenylation site was 3274 nucleotides downstream from ATG. Transfection experiments with chimeric luciferase constructs containing various truncated fragments of the 5'-region showed that the fragment -3160/+3 exhibited promoter activity in all tested cell lines and permitted the definition of a minimal 200-bp promoter (-603/-411) containing three putative Sp1-binding sites and two initiator elements. Transcriptional activity of this region was inhibited by the addition of mithramycin, a specific inhibitor of Sp1 binding to GC-rich sequences. The search for sequence variants within a fragment covering 1.7 kb of 5'-flanking region and the coding region allowed us to identify five novel single nucleotide polymorphisms. Interestingly, the G/C substitution at position -98 relative to the start codon was common and in complete linkage with a previously identified insertion/deletion polymorphism in the coding region which was showed to affect alpha2B-adrenergic receptor function. Based on transfection data and computer-assisted sequence analysis, the -98 G/C single nucleotide polymorphism was located within a portion of the 5'-UTR (-127/+3) affecting luciferase activity and it created additional putative binding site for Sp1. However, G/C substitution had no significant incidence on promoter activity in BHK-21 or HeLa cells.

We have investigated the mechanisms whereby alpha(2B)-adrenergic receptor (alpha(2B)-AR) promotes MAPK activation in a clone of the renal tubular cell line, LLC-PK1, transfected with the rat nonglycosylated alpha(2)-AR gene. Treatment of LLC-PK1-alpha(2B) with UK14304 or dexmedetomidine caused arachidonic acid (AA) release and ERK2 phosphorylation. AA release was abolished by prior treatment of the cells with pertussis toxin, quinacrine, or methyl arachidonyl fluorophosphonate but not by the addition of the MEK inhibitor U0126. The effects of alpha(2)-agonists on MAPK phosphorylation were mimicked by cell exposure to exogenous AA. On the other hand, quinacrine abolished the effects of UK14304, but not of AA, suggesting that AA released through PLA2 is responsible for MAPK activation by alpha(2B)-AR. The effects of alpha(2)-agonists or AA were PKC-independent and were attenuated by indomethacin and nordihydroguaiaretic acid. Treatment with batimastat, CRM 197, or tyrphostin AG1478 suppressed MAPK phosphorylation promoted by alpha(2)-agonist or AA. Furthermore, conditioned culture medium from UK14304-treated LLC-PK1-alpha(2B) induced MAPK phosphorylation in wild-type LLC-PK1. Based on these data, we propose a model whereby activation of MAPK by alpha(2B)-AR is mediated through stimulation of PLA2, AA release, generation of AA derivatives, activation of matrix metalloproteinases, release of heparin-binding EGF-like growth factor, transactivation of epidermal growth factor receptor, and recruitment of Shc. Whether this pathway is particular to alpha(2B)-AR and LLC-PK1 or whether it can be extended to other cell types and/or other G-protein-coupled receptors remains to be established.

In the rat proximal tubule, the alpha(2B)-adrenergic receptor (alpha(2B)-AR) enhances Na(+) reabsorption by increasing the activity of Na(+)/H(+) exchanger isoform NHE3. The mechanisms involved are unclear, and inhibition of cAMP production remains controversial. In this study, we reinvestigated alpha(2B)-AR signaling pathways using rat proximal tubule cells (PTC) in primary culture and LLC-PK(1) cells permanently transfected with the RNG gene (rat nonglycosylated alpha(2)-AR). Binding experiments indicated that PTC express substantial amounts of alpha(2B)-AR (130 fmol/mg protein), and only RNG transcripts were detected. In both cell types, the alpha(2B)-AR is coupled to G protein, and its stimulation by dexmedetomidine, but not by UK-14304, provoked a significant inhibition of the accumulation of cAMP induced by forskolin or parathyroid hormone. Exposure to alpha(2)-agonists increased arachidonic acid release and caused extracellular signal-regulated kinase (ERK)1/2 phosphorylation, which correlated with enhanced mitogen-activated protein kinse (MAPK) activity and nuclear translocation. MAPK phosphorylation was blunted by pertussis toxin but not by protein kinase C desensitization, and it coincided with transient phosphorylation of Shc. Finally, treatment with UK-14304 accelerated cell growth. Further studies will be necessary to clarify the precise mechanism of MAPK activation, but the present data suggest that alpha(2B)-AR may play a positive role during tubular regeneration.

1. Rat foetal liver contains large amounts of alpha2-adrenoceptors. The present work aimed to identify the receptor subtype and the cell type accounting for high expression and to clarify the mechanisms responsible for the sharp decrease in hepatic receptivity occurring during the late stage of foetal development. 2. Binding experiments indicated that the density of alpha2-adrenoceptors in the foetal liver (embryonic day 18; 615+/-155 fmol mg(-1) of protein) is 18 fold higher than in newborn or adult (35.2+/-4.3 fmol mg(-1)). A high amount of receptor is also found in the placenta (443+/-53 fmol mg(-1)). In both tissues, the rank order of antagonists to inhibit radioligand binding matched the pharmacological profile of the alpha2B-adrenoceptor and exclusively RNG transcripts were detected by RNase protection assays. 3. Isolation of cell fractions from foetal liver showed that alpha2B-adrenoceptor is primarily expressed by haematopoietic cells. Consistent with this view, the receptor is found to be abundant in foetal blood, carried by reticulocytes. The expression in blood gradually declines to zero at 3 weeks of age and it is not recovered following induction of reticulocytosis in adults. 4. In foetal reticulocytes, a low proportion of the receptor population is coupled to G-protein. The alpha2-agonist UK14304 has a marginal effect on cyclic AMP level but significantly increases arachidonic acid release. The function of the receptor remains to be elucidated. However, together with observations on alpha2B-knockout mice, the current finding strongly suggests a role for alpha2B-adrenoceptor during foetal haematopoiesis in rodents.

Hck, a protein-tyrosine kinase of phagocytes, is the unique member of the Src family expressed under two alternatively translated isoforms differing in their N-terminal site of acylation: p61(Hck) has an additional 21-amino acid sequence comprising a single myristoylation motif, whereas p59(Hck) N terminus has myristoylation and palmitoylation sites. To identify the molecular determinants involved in the targeting of each isoform, they were fused to GFP and expressed in HeLa and CHO cells. p61(Hck) was associated with lysosomal vesicles, whereas p59(Hck) was found at the plasma membrane and to a low extent associated with lysosomes. Their unique N-terminal domains were sufficient to target GFP to the corresponding intracellular compartments. Mutation of the palmitoylation site of p59(Hck) redirected this isoform to lysosomes, indicating that the palmitoylation state governs the association of p59(Hck) with the plasma membrane or with lysosomes. In addition, both isoforms and the nonpalmitoylated p59(Hck) mutant were found on the Golgi apparatus, suggesting a role of this organelle in the subcellular sorting of Hck isoforms. Regarding their subcellular localizations, we propose that bi-acylated p59(Hck) might transduce plasma membrane receptor signals, whereas p61(Hck) and the nonpalmitoylated p59(Hck) might control the biogenesis of phagolysosomes, two functions yet proposed for Hck in phagocytes.

The heterologous regulation of the alpha2C-adrenergic receptor (alpha2C-AR) was investigated in the HepG2 cell line. Binding of [(3)H]MK912 (alpha2-antagonist) to membranes from cells submitted to various treatments showed that exposure to insulin, phorbol 12-myristate 13-acetate, or dexamethasone did not affect receptor density. On the other hand, treatment with forskolin resulted in a large reduction of alpha2C-AR number. The effect of forskolin was mimicked by 8-br-cAMP and was abolished by the protein kinase A inhibitor, H89. The action of cAMP was slow (t(1/2) = 23 h), dose-dependent, and additive to the receptor down-regulation elicited by the alpha2-agonist, UK14304. Furthermore, the diminution of receptor was not caused by an increased rate of its degradation but resulted from a decrease in the steady state amounts of alpha2C4-mRNA. As assessed by experiments in the presence of actinomycin D, the stability of alpha2C4-mRNA was not affected by 8-br-cAMP or forskolin. By contrast, the activity of a luciferase construct containing the entire promoter region of the alpha2C4 gene (1.9 kilobase pairs) was inhibited, indicating that the primary mechanism of action of the two compounds is at the transcriptional level. Deletions in the 5'-end of this construct showed that the elements responsible for cAMP responsiveness lie within a 242-base-pair fragment of the gene promoter (nucleotides -236/+6 relative to transcription start). Band-shift experiments indicated that nuclear factors bind to this region in a cAMP-dependent manner. The determination of the actual cis- and trans-acting elements involved will be the object of future investigation, but the present study provides evidence for transcriptional regulation of human alpha2C-AR by cAMP.

1. Previous studies of the regulation of the alpha2C-adrenoceptor in OK and in transfected cells have led to discrepant conclusions. In the present work, we examined the homologous regulation of the human alpha2C-adrenoceptor in the hepatocarcinoma cell-line, HepG2; a model which expresses this subtype spontaneously. 2. Short-period treatment of the cells with UK14304 provoked neither a diminution of the potency of the alpha2-agonist to inhibit forskolin-induced cyclic AMP-accumulation nor a change in the degree of receptor coupling to G-proteins. 3. Long-period exposure to UK14304 resulted in a large reduction of [3H]MK912 binding sites (55% decrease). The action of UK14304 was dose-dependent (EC50 = 190 +/- 45 nM), rapid (t1/2 = 4.2 h) and reversible. Receptor down-regulation was also observed with clonidine or (-)adrenaline (38 and 36% decrease, respectively) and was blocked by the addition of alpha2-antagonists. 4. Conversely to that observed with alpha2-agonists, treatment of the cells with RX821002 or yohimbine alone, but not with phentolamine, promoted a significant increase of the receptor expression. 5. The observed alterations of receptor density are not the reflection of changes at the alpha2C4 mRNA level. Estimation of the receptor protein turnover and measurement of its half-life demonstrated that down-regulation by alpha2-agonists and up-regulation by alpha2-antagonists, with inverse-agonist efficacy, are respectively the consequence of increased and decreased rate of receptor degradation. 6. In conclusion, our data show that alpha2C-adrenoceptor does not undergo desensitization but is down-regulated in HepG2. The lack of desensitization agrees with previous results obtained in cells transfected with the alpha2C4 gene, but not with observations made in OK cells. Inversely, down-regulation fits with results obtained in OK but not in transfected cells. The reasons for these discrepancies are discussed. Our results also demonstrated that certain alpha2-antagonists behave as inverse agonist on the HepG2 model and thus provide for the first time evidence of inverse efficacy of antagonists on a cellular model expressing physiological level of a wild-type alpha2-adrenoceptor.

Screening of a human foetal brain genomic DNA library allowed us to isolate an EcoRI-EcoRI fragment containing 6 kb of the 5'-flanking region, the open reading frame and 4 kb of the 3'-flanking region of the alpha2C4 gene. Analysis of the sequenced region (4850 bp) revealed that the first 900 bp 5' to the start codon are very rich in GC (84%), contain several Sp1-binding sites and lack a consensus TATA box. The 5'- and 3'-ends of the alpha2C4 transcript were determined by RNase-protection assays carried out with a series of antisense probes. The data obtained with cellular RNA from HepG2 cells demonstrated that transcription is initiated 891 bases upstream of the translation-start site and that the polyadenylation site is located 550 bases downstream of the stop codon. These results are consistent with the existence of a non-conventional TATA box (TTAGAAA) and the presence of a unique polyadenylation signal (AATAAA). They also fit with the size of alpha2C4-RNA found by Northern-blot analysis (2.9 kb). The transcriptional activity of the alpha2C4 promoter region was investigated by transfecting several cell types with chimaeric constructs containing various fragments of the 5'-non-coding region and luciferase as a reporter gene. The activity of the construct containing the entire 5'-non-coding region appeared to depend on the host cell. Removal of the 5'-untranslated region resulted in loss of cell specificity and a concomitant increase in luciferase activity. Transfection of HepG2 and SK-N-MC cells with constructs deleted of additional 5'-flanking fragments permitted the definition of a minimal 200 bp promoter fragment containing the pseudo-TATA box and two putative SP1-binding sites.

Hormone-sensitive lipase (HSL) catalyses the rate-limiting step of adipose tissue lipolysis. The human HSL gene is composed of nine exons encoding the adipocyte form and a testis-specific coding exon. Northern blot analyses showed that human adipocytes express a 2.8 kb HSL mRNA, suggesting the presence of a short (20-150 bp) 5' untranslated region (5'-UTR). A single 5'-UTR of approx. 70 nt was detected in RNase H mapping experiments. Two 5'-UTRs of 70 and 170 nt respectively were obtained by rapid amplification of cDNA ends and cDNA library screenings. RNase protection experiments, with probes derived from the two products, showed that human adipocyte HSL mRNA contains only the 70 nt product. Primer extension analysis mapped the transcriptional start site 74 nt upstream of the start codon. In HT29, a human cell line expressing HSL, the presence of the short or the long 5'-UTR is mutually exclusive. The short and long 5'-UTR exons were located 1.5 and approx. 13 kb respectively upstream of the first coding exon. Various portions of the 5'-flanking region upstream of the short product exon were linked to the luciferase gene and transfected into cells that express HSL (HT29 cells and rat adipocytes) and do not express HSL (HeLa cells). High luciferase activity was found for constructs containing the sequence between nt -2400 and -86, but not for shorter constructs. An analysis of 14 kb of genomic sequence revealed the presence of five DNase I hypersensitive sites associated with active gene transcription. Three of the sites are located in the vicinity of the transcriptional start site and could be linked to the minimal promoter activity. Two of the sites are located downstream of the exon containing the start codon, suggesting the presence of intronic regulatory elements.

It is now clearly established that alpha-2 adrenergic receptors can be subdivided in three pharmacological subtypes (alpha-2A, alpha-2B and alpha-2C) encoded by distinct genes (alpha 2C10, alpha 2C2 and alpha 2C4, respectively, in humans). Whereas the study of the regulation of the human alpha-2A adrenergic receptor and of the promoter region of the alpha 2C10 gene has being greatly helped by the availability of the colon carcinoma cell line HT29, the study of the other human receptor subtypes has thus far been limited to homologous desensitization/down-regulation in transfected cells, because of the lack of human cellular models constitutively-expressing alpha-2B or alpha-2C adrenergic receptors. Several human cell lines were thus screened, in an attempt to find such models. Radioligand binding studies with [3H]RX821002 and [3H]MK912, reverse transcription-polymerase chain reactions and RNase mapping experiments with pairs of primers and riboprobes specific for each subtype demonstrated that the hepatoma cell line HepG2 and the neuroblastoma cell line SK-N-MC possess alpha-2 adrenergic receptors of the alpha-2C subtype. However, whereas HepG2 expresses exclusively alpha-2C receptors (55 +/- 7 fmol of [3H]MK912 binding sites/mg of protein), SK-N-MC expresses both alpha-2A and alpha-2C subtypes in fairly similar amounts (20 +/- 8 and 23 +/- 3 fmol of [3H]MK912 binding sites/mg of protein, respectively). The study of the inhibition of 3H-labeled antagonist binding by UK14304 demonstrated that a fraction of the receptor population was coupled to pertussis toxin-sensitive G-proteins, which were identified as Gi2 and Gi3 by immunoblotting. The alpha-2 agonist was, moreover, able to decrease forskolin-stimulated cAMP production by 47% in HepG2 and 23% in SK-N-MC, demonstrating that inhibition of adenylyl cyclase is one of the primary mechanisms of signal transduction in both cell lines. HepG2 and SK-N-MC are the first human cell lines unquestionably shown to natively express alpha-2C adrenergic receptors. The discovery of these two models may be useful for future study of the regulation of alpha 2C4 gene expression in cells of different origins and investigation of the reciprocal regulation of alpha-2A and alpha-2C subtype in single cells.

This study was undertaken to investigate the consequences of diabetes on Gi-protein expression and alpha 2-adrenergic receptivity in rat intestinal mucosa. Experimental diabetes was induced by treatment with streptozotocin. Quantification of alpha i-subunits by immunoblotting demonstrated that the level of the G alpha i2 but not the G alpha i3 subunit was markedly decreased in jejunum and colon membranes from diabetic rats as compared to controls. Parallel assessment of sympathetic innervation was performed by determination of norepinephrine content, measurement of tyrosine hydroxylase and monoamine oxidase activities, and quantification of alpha 2-adrenergic receptors in the different segments. At this stage of diabetes (6 weeks after streptozotocin injection), none of these parameters was significantly modified. Consequently, the decrease in G alpha i2 amount appears to be independent of the neuropathy describe in later stages of diabetes.

Previous studies on the intestinal epithelium from various species have shown that the number of alpha 2-adrenergic receptors in immature cells from the crypts is several-fold higher than in mature cells from the villi, thus suggesting an inverse relationship between enterocytic differentiation and the expression of this inhibitory receptor. The receptor density along the surface-crypt axis of the human colonic mucosa is correlated with the amount of alpha 2C10 mRNA; however, the mechanisms underlying this regulation remain unknown. The human colonic adenocarcinoma cell line HT29, which expresses the alpha 2A-adrenergic receptor and is able to undergo enterocytic differentiation, is a suitable model with which to investigate this question in vitro. In this study, we explored the effects of short chain fatty acids (SCFAs), differentiating agents normally present in the colon lumen, on alpha 2-adrenergic receptor expression. Exposure of HT29 cells to butyrate and propionate, but not acetate, resulted in a large diminution of [3H]RX821002 binding sites. The reduction of alpha 2-adrenergic receptor number induced by butyrate or propionate was due to decreased amounts of alpha 2C10 mRNA and was associated with an increase of alkaline phosphatase activity, which reflected the emergence of a more differentiated phenotype. The changes in alpha 2C10 mRNA level induced by both SCFAs were dose-dependent, rapid, and reversible and resulted from a diminution in the transcription rate of the alpha 2C10 gene. Finally, these effects were mimicked by trichostatin A, indicating that they are triggered primarily through inhibition of histone deacetylases. The present findings demonstrate that decrease of alpha 2-adrenergic receptor expression is a very early event of the HT29 cell differentiation process. They also suggest that SCFAs, which originate from bacterial fermentation of dietary fibers, may play a role in the regulation of the alpha 2-adrenergic receptivity of colonic mucosa in vivo.