Combining Cadherin Expression with Molecular Markers Discriminates Invasiveness in Growth Hormone and Prolactin Pituitary Adenomas.

Although growth hormone (GH)- and prolactin (PRL)-secreting pituitary adenomas are considered benign, in many patients, tumour growth and/or invasion constitute a particular challenge. In other tumours, progression relies in part on dysfunction of intercellular adhesion mediated by the large family of cadherins. In the present study, we have explored the contribution of cadherins in GH and PRL adenoma pathogenesis, and evaluated whether this class of adherence molecules was related to tumour invasiveness. We have first established, by quantitative polymerase chain reaction and immunohistochemistry, the expression profile of classical cadherins in the normal human pituitary gland. We show that the cadherin repertoire is restricted and cell-type specific. Somatotrophs and lactotrophs express mainly E-cadherin and cadherin 18, whereas N-cadherin is present in the other endocrine cell types. This repertoire undergoes major differential modification in GH and PRL tumours: E-cadherin is significantly reduced in invasive GH adenomas, and this loss is associated with a cytoplasmic relocalisation of cadherin 18 and catenins. In invasive prolactinomas, E-cadherin distribution is altered and is accompanied by a mislocalisation of cadherin 18, ß-catenin and p120 catenin. Strikingly, de novo expression of N-cadherin is present in a subset of adenomas and cells exhibit a mesenchymal phenotype exclusively in invasive tumours. Binary tree analysis, performed by combining the cadherin repertoire with the expression of a subset of known molecular markers, shows that cadherin/catenin complexes play a significant role in discrimination of tumour invasion.

Gap junctions mediate electrical signaling and ensuing cytosolic Ca2+ increases between chromaffin cells in adrenal slices: A role in catecholamine release.

In adrenal chromaffin cells, a rise in cytosolic calcium concentration ([Ca(2+)]i) is a key event in the triggering of catecholamine exocytosis after splanchnic nerve activation. Action potential- or nicotine-induced [Ca(2+)]i transients are well described in individual chromaffin cells, but whether they remain spatially confined to the stimulated cell or propagate to adjacent cells is not yet known. To address this issue, the spatiotemporal organization of electrical and associated Ca(2+) events between chromaffin cells was investigated using the patch-clamp technique and real-time confocal imaging in rat acute adrenal slices. Spontaneous or electrically evoked action potential-driven [Ca(2+)]i transients were simultaneously detected in neighboring cells. This was likely attributable to gap junction-mediated electrotonic communication, as shown by (1) the bidirectional reflection of voltage changes monitored between cell pairs, (2) Lucifer yellow (LY) diffusion between cells exhibiting spontaneous synchronized [Ca(2+)]i transients, and (3) the reduction of LY diffusion using the uncoupling agent carbenoxolone. Furthermore, transcripts encoding two connexins (Cx36 and Cx43) were found in single chromaffin cells. This gap junctional coupling was activated after a synaptic-like application of nicotine that mediated synchronous multicellular [Ca(2+)]i increases. In addition, nicotinic stimulation of a single cell triggered catecholamine release in coupled cells, as shown by amperometric detection of secretory events. Functional coupling between chromaffin cells in situ may represent an efficient complement to synaptic transmission to amplify catecholamine release after synaptic stimulation of a single excited chromaffin cell.

Synthesis, conformational studies and biological activity of N(alpha)-mono-biotinylated rat relaxin.

Biotin-avidin immobilization can be a useful tool in structure-function studies of hormone receptors. A crucial step is the preparation of a specifically biotinylated hormone that is able to bind to its receptor while leaving the biotin group free for interaction with avidin. The receptor for relaxin, an ovarian peptidic hormone produced during pregnancy, has not yet been isolated. We therefore undertook to prepare a specifically monobiotinylated rat relaxin for use in ligand-searching strategies. Rat relaxin is a convenient analogue because reliable bioassays exist, thus allowing assessment of the effect of N-biotinylation on bioactivity. To help improve the yield of the two-chain, three-disulfide bond rat relaxin, 2-hydroxy-4-methoxybenzyl (Hmb) backbone protection was used during the solid-phase assembly of the B-chain to help prevent any possible chain aggregation. As a final step, while the protected peptide was still on the resin, the biotin label was introduced at the N-terminus of the B-chain using standard coupling protocols. The chain combination with the A-chain was accomplished in reasonable yield. Secondary structural measurements demonstrated that the biotin caused the starting B-chain to adopt a more ordered conformation. The labelled synthetic relaxin exhibited similar circular dichroism spectra to native and synthetic single B-chain peptides. In addition, the biotinylated relaxin showed no significant difference in its chronotropic activity in the rat isolated heart assay compared with the native peptide. Biosensor studies showed that antibody recognition was retained upon attachment of the synthetic relaxin to the streptavidin-derivatized surface.

The human neuroblastoma SK-SY5Y cell line bears functional endothelin-A-receptors and endothelin.

The study reported here characterizes the presence both of endothelin (ET) receptors and of a synthesizing ET apparatus in the human neuroblastoma SK-SY5Y cell line. We demonstrated, using reverse transcriptase polymerase chain reaction (RT-PCR), that these cells bound [125I]ET-1. The potency order of ET analogs to inhibit [125I]ET-1 binding was consistent with the presence of ET(A)-receptors. [Ca2+]i was increased by both ET-1 and ET-3 (potency order: ET-1 > ET-3. The mRNAs of preproendothelin-1 and of endothelin converting enzyme (ECE) were expressed by cells, as shown by RT-PCR studies. These mRNAs were translated into functional proteins as the cells were able to release mature (1-21) ET-like immunoreactivity into the culture medium. That secretion was time-dependent and was enhanced by treatment of the cells by the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate. These results show that the human SK-SY5Y neuroblastoma cell line produces mature ET which could act as an autocrine/paracrine factor these cells.

Novel strategy for the synthesis of template-assembled analogues of rat relaxin.

The 'template-assembled synthetic protein' (TASP) concept provides a simple and elegant approach for the preparation of analogues that retain key structural elements. We have synthesized TASP molecules containing the putative active site of relaxin, a peptide that has similar structural features to insulin but a markedly different biological role. Two types of chemoselective thiol ligation strategies (thioether and thiazolidine) were used and compared. The synthetic pendant peptides contain an essential region for bioactivity that is located in the alpha-helical region of the relaxin B-chain. Depending on whether the thioether or the thiazolidine chemistry was used to attach the peptides to the template, the reacting amino acid was placed either at the C-terminus or N-terminus, respectively, thus allowing the choice of orientation relative to the carrier molecule. The template molecule consists of a decapeptide with two proline-glycine turns and four evenly spaced lysine residues that were functionalized with the appropriate chemical moiety. This allowed reaction with the appropriately derivatized peptides in solution. To improve the template ligation step using the thioether approach, a pendant peptide C-terminal cysteamine residue was used to reduce potential steric hindrance during conjugation. The design of the peptides as well as the synthetic strategy resulted in the acquisition of mimetics showing weak non-competitive and weak competitive antagonist properties.

Antiproliferative, differentiating and apoptotic effects elicited by imidazo[1,2-a]pyrazine derivatives.

1. The activity of two series of imidazo[1,2-a]pyrazine derivatives on cell proliferation and differentiation and on apoptosis was examined in relation to their effects on phosphodiesterase (PDE) activity and on purinoceptors. 2. In the first series SC-18 and SC-51 inhibited mitogen-induced 3H-thymidine incorporation in human lymphocytes. 3. The compounds of the new series PAB13, PAB23 and SCA40 inhibited the proliferation of the HEL cell line. 4. Nine imidazo[1,2-a]pyrazine derivatives of the new series have been studied on the Dami cell proliferation. SCA41 and SCA44 inhibited cell growth, SCA40 and PAB40 were moderately effective, whereas PAB12 and PAB30 were devoid of effect. The antiproliferative effects of these six non-cytotoxic compounds could not be related to their action on PDE or on purinoceptors, but rather to their lipophilicity. Conversely, for PAB13, PAB15, and PAB23, the decrease in cell number was related to their cytotoxic and apoptotic effects through their cAMP-increasing and PDE-inhibitory potency, but unrelated to an effect on purinoceptors. 5. Imidazo[1,2-a]pyrazine derivatives decreased the expression of Glycoprotein (GP)Ib in Dami cells while some of them enhanced that of GPIIb/IIIa. These effects appeared to involve inhibition of both cAMP- and cGMP-PDE. 6. These studies demonstrate the potential interest of imidazo[1,2-a]pyrazine derivatives in the query of novel anticancer drugs.

Change of endothelin receptor subtype in the MEG-01 human megakaryoblastic cell line.

The aim of this investigation was to determine whether the endothelin receptor subtype of a megakaryoblastic cell line (MEG-01) changes during culture passages as cells undergo maturation and differentiation. On early-passage cells, binding of [125I]endothelin-1 was completely inhibited by 1 microM BQ 123 (cyclo-[D-tryptophanyl-D-aspartyl-prolyl-D-valyl-leucyl]), but not by sarafotoxin 6C. Also the endothelin-1-enhancing effect on [Ca2+]i was prevented by BQ 123, whereas sarafotoxin 6C had no effect on [Ca2+]i. In late-passage cells, endothelin ET(B) analogs, unlike endothelin ET(A) analogs, competed with binding of [125I]endothelin-1. Endothelin ET(B) receptor agonists increased [Ca2+]i while the endothelin-1-induced response was inhibited by BQ 788 ([N-[(2R,6S)-2,6-dimethyl-piperidinocarbonyl]-4-methyl-D-leucyl]-[ N(omega)-(methoxycarbonyl)-D-tryptophanyl]-D-norleucine), but not by BQ 123, although both endothelin ET(A) and ET(B) receptor mRNAs were expressed, as shown by reverse transcriptase-polymerase chain reaction. These results demonstrate that in MEG-01 cells switch from expression of endothelin ET(A) to expression of ET(B) receptors during culture. The data also suggest that late-passage MEG-01 cells look like platelets, in terms of endothelin receptor subtype.

Signaling mechanisms induced by endothelin agonists in human adrenal glomerulosa cells.

Endothelin-1 (ET-1) exhibits secretagogue and trophic actions on the adrenal zona glomerulosa (ZG). Little information is available on the intracellular signaling events that follow stimulation of ET receptors on ZG cells. This study examined the expression of ET receptor subtypes and their involvement in transduction mechanisms induced by ET agonists on human ZG cells in primary culture. RT-PCR allowed the detection of both ETA and ETB receptor mRNAs in these cells. ET-1 induced a concentration-dependent increase in inositol phosphate (IP) accumulation in the presence of LiCl, whereas ETB agonists were inactive. The ET-1-induced increase in IP accumulation was prevented by BQ-123. ET-1 evoked an increase in [Ca2+]i, which was partially prevented by BQ-788. IRL 1620 also delayed the rise in [Ca2+]i. These results show that in human adrenal ZG cells, ET-1 induces an increase in IP accumulation through ETA receptor activation and evokes a rise in [Ca2+]i via stimulation of both ETA and ETB receptors.

Human bone marrow platelet progenitors express and release endothelin-1.

Because it has been shown that human platelet precursors from normal bone marrow express preproendothelin-1 (ET-1) mRNA, this investigation was designed to find out whether these cells could synthesize and release mature ET-1 and express ET receptors. Therefore, we examined the expression of endothelin-converting enzyme (ECE) mRNA and of mRNAs for ET receptors in cells purified from normal bone marrow and measured immunoreactive (ir)ET in their culture medium. RT-PCR applied to RNAs from platelet precursors yielded amplified fragments of the expected sizes of 567, 428, and 299 bp for ECE, ETB and ETA receptors, respectively. These cells released ir-ET into the culture medium in a time-dependent fashion. These results raise the possibility of autocrine actions of the intrinsic ET system in bone marrow platelet precursor cells.

Phosphoramidon-sensitive and -insensitive endothelin-converting enzyme in human megakaryoblastic cell lines.

The human megakaryoblastic cell lines HEL, MEG-01, and DAMI express preproendothelin-1 mRNA. This investigation was designed to find out whether they could also express endothelin-converting enzyme (ECE) and release mature endothelin (ET). RT-PCR applied to RNA isolated from the cell lines amplified fragments of the expected size. The amplified cDNA of MEG-01 was submitted to restriction enzymes, which generated the expected subfragments. Membrane ECE activity was phosphoramidon-sensitive, in contrast to the cytosolic activity capable of producing ET-1 from big ET-1. The three cell lines produced ir-ET in a time-dependent manner. These results show that human megakaryoblastic cell lines express functional, phosphoramidon-sensitive and insensitive ECE activity and produce mature ET.

Adrenal steroid-secreting cells express endothelin-1.

Endothelin-1 (ET-1) is involved in adrenal steroid secretion but its cell origin remains unclear. We showed, using RT-PCR the expression of the mRNAs for preproET-1 and ECE-1 in primary cultures of human adrenal cells enriched in glomerulosa cells. Since these expressions could be due to contamination of steroid secreting cells by other cells, we also used the human adrenocortical cell line H295R, which was shown to produce steroids. This cell line also expressed preproET-1-RNA and released mature ET. Functional ET receptors were shown on H295R and cultured human adrenocortical cells. These findings indicate that adrenal steroid-secreting cells synthesize and release ET-1, raising the possibility for an autocrine-paracrine effect of ET-1 on adrenocortical functions.

Apoptotic effects of imidazo[1,2-a]pyrazine derivatives in the human Dami cell line.

cAMP-elevating agents like phosphodiesterase inhibitors and purines have been shown to induce apoptosis. In the present work we have studied the effects of imidazo[1,2-a]pyrazine derivatives with a purine-like structure: PAB13 (6-bromo-8-(methylamino)imidazo[1,2-a] pyrazine), PAB15 (6-bromo-8-(ethylamino)imidazo[1,2-a]pyrazine), PAB23 (3-bromo-8-(methylamino)imidazo[1,2-a]pyrazine) on the growth of the Dami cell line in comparison to that of adenosine. The growth effect of PAB13, PAB15 and PAB23 was investigated in relation to their phosphodiesterase-inhibitory action and their activity on purinoceptors. Inhibition in cell growth was up to 71.0%, 76.3% and 89.7% for PAB23, PAB13 and PAB15, respectively and 100% for adenosine. Cell viability was affected in a concentration-dependent manner by PAB13, PAB15 and adenosine, with a correlation between growth inhibition and cytotoxicity. These effects of imidazo[1,2-a]pyrazine derivatives were found to be unrelated to an action on purinoceptors, but rather appear quantitatively linked to their ability in inducing apoptosis through their cAMP-increasing and phosphodiesterase-inhibitory potency.

Endothelin expression in human megakaryoblastic leukemia cell lines and normal platelet precursors.

The aim of the present investigation was to determine whether endothelin (ET) could be expressed in and released from the human leukemia megakaryoblastic cell lines HEL, MEG-01, DAMI and the normal human platelet progenitors. Using the reverse transcriptase-polymerase chain reaction (RT-PCR) on total RNA isolated from the cells, we amplified a cDNA of the expected size (453 bp). Southern-blotting hybridization revealed that RT-PCR products from the cell lines were specific of ET-1 mRNA. Immunocytochemical analyses highlighted immunoreactive ET-1 in the cytoplasm of these cells which also released the mature peptide. ET-1 release from the three cell lines was increased by thrombin exposure. Although MEG-01 cells express ET receptors, ET-1, the selective ETB agonist sarafotoxin 6C and the non-selective ET-receptor antagonist PD 142893 showed no proliferative or antiproliferative action in basal or stimulating medium. This indicated a lack of autocrine ET-mediated effect on growth. These results demonstrate for the first time that human megakaryoblastic leukemia cell lines and normal bone marrow platelet precursors express ET-1 mRNA and release the mature peptide.

Effect of chronic treatment with trandolapril or enalapril on brain ACE activity in spontaneously hypertensive rats.

The aim of the present study was to determine whether the new ACE inhibitor trandolapril was able to inhibit brain ACE activity in spontaneously hypertensive rats (SHRs). Therefore, we have measured ex vivo ACE activity in discrete brain areas of SHRs after a 2-week oral treatment with trandolapril (0.001, 0.01, 0.1 and 1 mg/kg/day). The effects of trandolapril were compared to those of enalapril (10 mg/kg/day), used as a reference compound. Enalapril induced a decrease in ACE activity in brain areas not protected by the blood brain barrier (subfornical organ and median eminence) and in cerebral cortex. Conversely, trandolapril at a dose of 0.01 mg/kg/day and above induced a dose-dependent inhibition of ACE activity in all brain areas assayed, including the supraoptic and paraventricular hypothalamic nuclei, septum, amygdala, hippocampus, cerebellar and cerebral cortex, nucleus of the tractus solitary and caudate nucleus. The inhibition was roughly similar in all brain areas studied. These data suggest that after chronic oral administration in SHRs, trandolapril or its metabolite, in contrast to enalapril or enalaprilat, was able to reach all brain areas of SHRs, including those protected by the blood brain barrier.

Expression of endothelin and ETB receptors in the megakaryoblastic MEG-01 cell line.

The presence of endothelin (ET) receptors and the nature of the subtype and expression of ET were investigated in the human megakaryoblastic cell line MEG-01. By the RT-PCR procedure, we have shown that both ETA and ETB receptor subtype mRNAs are expressed in the cells. However, binding experiments have shown that the selective ETB receptor antagonist BQ788, but not the selective ETA receptor antagonist BQ123, competes with the specific binding of [125I]ET-1. Using immunocytochemistry, RIA, and RT-PCR Southern blot, we have shown that MEG-01 cells express ET-1. In addition, ET (1-21)-like immunoreactivity was released from the cells into the culture medium, and this release was modulated by thrombin. These data suggest that an ET-1-mediated autocrine loop could occur in the human megakaryoblastic MEG-01 cell line.

Endothelin-1 and ETA receptor subtype are expressed in the gastric HGT-1 cell line.

Recent studies have revealed that endothelin-1 (ET-1) may be produced by human cancer cell lines and have suggested that in vivo the peptide might play a modulatory role in the growth of stromal cells surrounding tumor cells and/or in the growth of the cancer cells themselves, through paracrine or autocrine mechanisms. Therefore, we investigated whether ET-1 and ET receptors could be expressed in the human gastric cancer cell line HGT-1. By applying the reverse transcriptase polymerase chain reaction (RT-PCR) to total RNA extracted from the cells, using oligonucleotides synthesized from the sequence of the prepro-ET-1 mRNA, we have amplified a cDNA at the expected size (453 bp), which hybridized with a labeled ET-1-specific probe. In addition, RT-PCR was carried out to test whether HGT-1 cells expressed mRNA for ETA and/or ETB receptor subtypes. The amplified products of cDNA were at the size predicted for the ETA receptor (368 bp), whereas no ETB receptor mRNA could be detected.

Compared properties of trandolapril, enalapril, and their diacid metabolites.

The effects of 14-day trandolapril or enalapril treatment of spontaneously hypertensive rats (SHRs) were studied on blood pressure and angiotensin-converting enzyme (ACE) activity measured ex vivo in various organs. Both ACE inhibitors caused dose-dependent decreases in blood pressure and ACE activity, trandolapril being 30- and 400- to 1,000-fold more active than enalapril on blood pressure and ACE activity, respectively. However, comparison of ACE inhibitory activities of the diacid forms of trandolapril and enalapril, i.e., trandolaprilat and enalaprilat, measured in vitro on various tissues, showed that trandolaprilat was only three- to fivefold more active than enalaprilat. To understand the reasons for such discrepancies between ex vivo effects of ACE inhibitors and in vitro actions of their diacid metabolites, we measured the lipophilicities of the compounds and investigated the possibility that trandolapril could display an ACE inhibitory effect by itself. Trandolaprilat was found to be far more lipophilic than enalaprilat, as shown by reverse-phase high-performance liquid chromatography studies performed at pH 7.4 (log kw7.4 = 1.487 vs. 0.108). In addition, trandolapril was practically as active in vitro as its diacid metabolite (IC50 = 2.5 vs. 1.35 nM) in inhibiting ACE activity in the aorta, whereas enalapril was practically devoid of any effect (IC50 = 240 nM). Measurements of relative affinities of inhibitors or metabolites for purified human renal ACE showed that trandolapril displayed about 20% of the affinity of its diacid metabolite (IC50 = 15 vs. 3.2 nM); enalaprilat affinity (34 nM) was within the same range as those of trandolapril and trandolaprilat, whereas enalapril displayed a very low affinity for the purified enzyme (IC50 = 50 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of endothelins on the human megakaryoblastic cell line MEG-01.

Some effects of endothelin-1 (ET-1) were studied on the megakaryoblastic cell line MEG-01. ET-1 induced an elevation of the intracellular levels of Ca2+([Ca2+]i) as measured with the fluorescent indicator indo-1, which consists of an initial transient increase and an ensuing sustained plateau. The plateau phase was abolished by removal of extracellular Ca2+. In addition, ET-1 induced a rapid (within 5 s) increase in the accumulation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and more delayed increases in Ins(1,3,4)P3 and Ins(1,3,4,5)P4, but did not modify cAMP levels. ET-1 homologues (ET-2, ET-3, sarafotoxin 6b and vasointestinal constrictor) also induced biphasic effects on [Ca2+]i. The Ca2+ elevation was concentration dependent, the order of potency being sarafotoxin 6b > ET-1 > ET-2 = vasointestinal constrictor > ET-3. The actions of ET analogs in raising [Ca2+]i were mutually exclusive, suggesting that they act through the same mechanism. These results suggest that cells of the megakaryoblast/megakaryocyte lineage are targets for endothelins.

Platelet receptor expression on three human megakaryoblast-like cell lines.

Platelets, the progeny of bone marrow megakaryocytes, are nonnucleated cells; many platelet proteins, including platelet membrane receptors, are believed to be derived from megakaryocytes. Several hematopoietic cell lines that exhibit megakaryocytic characteristics have been established as models for the study of megakaryocyte biology. We report here the screening of platelet receptor expression, in terms of functional coupling with the formation of two second messengers, calcium and cAMP, in three cell lines exhibiting megakaryoblastic properties: HEL, MEG-01, and DAMI. We show that all these cell lines respond to thrombin, ADP, epinephrine, and prostaglandin E1 (PGE1). However, transmembrane signaling pathways appear partly different from those present in mature platelets, because the action of thrombin was found to be positively coupled with the cAMP pathway, in addition to that of calcium, and because PGE1, which interacts with the cAMP pathway, also raises intracellular calcium levels in the three cell lines studied. Furthermore, an endothelin-1-induced increase in intracellular calcium level was observed in MEG-01 cells, strongly suggesting the expression of endothelin receptors on platelet precursors cells, whereas the presence of such receptors is controversial on platelets. These cell lines should prove useful in further studies of the expression and molecular pharmacology of platelet receptors on platelet precursor cells, as well as for the investigation of functional roles for platelet receptors on megakaryoblastic cells.

Thrombin inhibits proliferation of the human megakaryoblastic MEG-01 cell line: a possible involvement of a cyclic-AMP dependent mechanism.

Thrombin, a potent platelet activating agent, has previously been found to increase intracellular calcium levels and/or thromboxane A2 synthesis in leukemic cell lines exhibiting specific markers of the megakaryocyte/platelet lineage. However, its functional role on these cells has not been defined. As thrombin is implicated in the regulation of cellular proliferation or differentiation in various other cell types, we investigated the functional effects of thrombin on the megakaryoblastic MEG-01 cell line, and further explored its receptor coupling mechanisms on these cells. We observed that thrombin caused in 1% serum containing culture medium, a reduction in the proliferation of MEG-01 cells, without affecting their differentiation stage as determined by the expression of platelet glycoproteins GPIIb/IIIa and GPIb, FVIII-related-antigen and cell-size measurement, which are specific markers for megakaryocyte maturation. In addition, incubation of MEG-01 cells with thrombin resulted in dose-dependent increases in cAMP levels, and in inositol-trisphosphate formation and intracellular Ca2+ levels. All these responses required thrombin proteolytic activity. The lipoxygenase inhibitor, nordihydroguaiaretic acid, blunted thrombin-induced calcium increase without affecting thrombin-induced increase in cAMP levels, suggesting different thrombin coupling mechanisms with these two second messenger pathways. In addition, the inhibitory effect of thrombin on MEG-01 cell growth was mimicked by cAMP level enhancing agents such as forskolin, prostaglandin E1 and Bt2cAMP. These results suggest the involvement of a cAMP-dependent mechanism in the thrombin-induced reduction in MEG-01 cell growth.