Role of serotonin 5-HT2A receptors in the development of cardiac hypertrophy in response to aortic constriction in mice.

Serotonin, in addition to its fundamental role as a neurotransmitter, plays a critical role in the cardiovascular system, where it is thought to be involved in the development of cardiac hypertrophy and failure. Indeed, we recently found that mice with deletion of monoamine oxidase A had enhanced levels of blood and cardiac 5-HT, which contributed to exacerbation of hypertrophy in a model of experimental pressure overload. 5-HT2A receptors are expressed in the heart and mediate a hypertrophic response to 5-HT in cardiac cells. However, their role in cardiac remodeling in vivo and the signaling pathways associated are not well understood. In the present study, we evaluated the effect of a selective 5-HT2A receptor antagonist, M100907, on the development of cardiac hypertrophy induced by transverse aortic constriction (TAC). Cardiac 5-HT2A receptor expression was transiently increased after TAC, and was recapitulated in cardiomyocytes, as observed with 5-HT2A in situ labeling by immunohistochemistry. Selective blockade of 5-HT2A receptors prevented the development of cardiac hypertrophy, as measured by echocardiography, cardiomyocyte area and heart weight-to-body weight ratio. Interestingly, activation of calmodulin kinase (CamKII), which is a core mechanism in cardiac hypertrophy, was reduced in cardiac samples from M100907-treated TAC mice compared to vehicle-treated mice. In addition, phosphorylation of histone deacetylase 4 (HDAC4), a downstream partner of CamKII was significantly diminished in M100907-treated TAC mice. Thus, our results show that selective blockade of 5-HT2A receptors has beneficial effect in the development of cardiac hypertrophy through inhibition of the CamKII/HDAC4 pathway.

High level of alpha2-adrenoceptor in rat foetal liver and placenta is due to alpha2B-subtype expression in haematopoietic cells of the erythrocyte lineage.

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.

Transcriptional down-regulation of the human alpha2C-adrenergic receptor by cAMP.

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.

Lack of palmitoylation redirects p59Hck from the plasma membrane to p61Hck-positive lysosomes.

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.

Alpha(2) adrenoceptors regulate proliferation of human intestinal epithelial cells.

BACKGROUND AND AIMS: Previous studies on rodents have suggested that catecholamines stimulate proliferation of the intestinal epithelium through activation of alpha(2) adrenoceptors located on crypt cells. The occurrence of this effect awaits demonstration in humans and the molecular mechanisms involved have not yet been elucidated. Here, we examined the effect of alpha(2) agonists on a clone of Caco2 cells expressing the human alpha(2A) adrenoceptor. METHODS: Cells were transfected with a bicistronic plasmid containing the alpha2C10 and neomycin phosphotransferase genes. G418 resistant clones were assayed for receptor expression using radioligand binding. Receptor functionality was assessed by testing its ability to couple Gi proteins and to inhibit cAMP production. Mitogen activated protein kinase (MAPK) phosphorylation was followed by western blot, and cell proliferation was estimated by measuring protein and DNA content. RESULTS: Permanent transfection of Caco2 cells allowed us to obtain a clone (Caco2-3B) expressing alpha(2A) adrenoceptors at a density similar to that found in normal human intestinal epithelium. Caco2-3B retained morphological features and brush border enzyme expression characteristic of enterocytic differentiation. The receptor was coupled to Gi2/Gi3 proteins and its stimulation caused marked diminution of forskolin induced cAMP production. Treatment of Caco2-3B with UK14304 (alpha(2) agonist) induced a rapid increase in the phosphorylation state of MAPK, extracellular regulated protein kinase 1 (Erk1), and 2 (Erk2). This event was totally abolished in pertussis toxin treated cells and in the presence of kinase inhibitors (genistein or PD98059). It was unaffected by protein kinase C downregulation but correlated with a transient increase in Shc tyrosine phosphorylation. Finally, sustained exposure of Caco2-3B to UK14304 resulted in modest but significant acceleration of cell proliferation. None of these effects was observed in the parental cell line Caco2. CONCLUSION: The results obtained in the present study support a regulatory role for alpha(2) adrenoceptors in intestinal cell proliferation.

Homologous regulation of the alpha2C-adrenoceptor subtype in human hepatocarcinoma, HepG2.

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.

Hsp72 mRNA production in cultured human cells submitted to nonlethal aggression by heat, ethanol, or propanol. Application to the detection of low concentrations of chromium(VI) (potassium dichromate).

The HT29 and HepG2 human cell lines have been shown to express stress proteins (heat shock proteins, HSP) when submitted to a variety of sublethal environmental aggressions. In the present study, these cells were submitted to standardized mild aggression by heat, ethanol, or propan-1-ol in vitro. Subsequent formation of the hsp72 mRNA was measured by a very specific RNase protection method using a radiolabeled antisense RNA probe. The accumulation of the mRNA coding for the HSP72 stress proteins was found to be maximum within 3 h after the aggression. Results were obtained faster and were much more interpretable than those from the classical method involving the autoradiography of electrophoretically separated 35S-labeled proteins, especially in the case of very weak, threshold-level, aggressions. When this model was used as a biological system for the detection of low concentrations of chromium(VI) (Cr2O7(2-)), it was possible to detect concentrations as low as 0.5 mumol/L. This indicates that measuring indices of stress induction in human cultured cells can be several orders of magnitude more sensitive than the commercial Microtox assay used for detecting low levels of pollution.

HepG2 and SK-N-MC: two human models to study alpha-2 adrenergic receptors of the alpha-2C subtype.

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.

Molecular cloning, sequencing and functional study of the promoter region of the human alpha2C4-adrenergic receptor gene.

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.

Characterization of the promoter of human adipocyte hormone-sensitive lipase.

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.

Experimental diabetes induces an early change in the level of the G-protein subunit, alpha i2, in rat intestinal mucosa.

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.

Regulation of alpha 2A-adrenergic receptor expression in the human colon carcinoma cell line HT29: SCFA-induced enterocytic differentiation results in an inhibition of alpha 2C10 gene transcription.

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.

Identification and subcellular distribution of the Gi-proteins in the enterocytic-differentiated adenocarcinoma cell-line, Caco-2.

As evidenced by pertussis toxin-catalysed [32P]ADP-ribosylation, immunoblotting and Northern blot, the human adenocarcinoma intestinal cell line Caco-2 expresses Gi2 and Gi3 proteins. The localization of these two Gis within the cell was investigated by using subcellular fractionation and confocal microscopy on intact cell layer. A brush-border rich fraction and a pellet containing the remaining cellular membranes were prepared. [32P]ADP-ribosylation and immunoblotting demonstrated the presence of both alpha i2 and alpha i3 in these two preparations. Immunofluorescence studies performed on intact cells grown on Transwell filters and viewed by confocal microscopy further confirmed the localization of alpha i3-subunit on basolateral as well as on apical membranes. In contrast, alpha i2-subunit was shown to accumulate mainly in the intra-cellular compartment while only faint staining of the plasma membrane was detectable. Based upon double-labelling experiments with antibody against rough endoplasmic reticulum (RER), there is a strong possibility that intra-cellular sites of alpha i2-subunit correspond to association with RER membranes.