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.

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.

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.

Prejunctional actions of piribedil on the isolated kidney of the rabbit: comparison with apomorphine.

1 The effects of piribedil on contractile responses and noradrenaline release evoked by sympathetic nerve stimulation have been studied in the isolated kidney of the rabbit. These effects were compared to those of apomorphine.2 Electrical stimulation (2, 5 and 10 Hz) of sympathetic renal nerves produced frequency-dependent increases in perfusion pressure and noradrenaline release. Piribedil did not affect (0.1 mug/min) or diminished (1 and 10 mug/min) the stimulation-evoked increase in perfusion pressure, and increased noradrenaline release in a dose-dependent manner.3 Increases in renal perfusion pressure and noradrenaline release induced by electrical stimulation were decreased by apomorphine (0.1 and 1 mug/min). These inhibitory effects were more marked at low frequencies of stimulation and were prevented by haloperidol (0.2 mumol/1).4 Piribedil (0.1 and 1 mug/min) and apomorphine (0.1, 1 and 10 mug/min) did not affect the increases in renal perfusion pressure elicited by exogenously administered noradrenaline, but piribedil (10 mug/min) diminished them.5 In the presence of desipramine (0.5 mumol/l), piribedil (0.1, 1 and 10 mug/min) produced a dosedependent inhibition of the increases in renal perfusion pressure and noradrenaline release evoked by sympathetic nerve stimulation; the inhibitory effect of piribedil was more marked at low frequencies of stimulation and was prevented by haloperidol.6 Piribedil increased the resting release of noradrenaline from the rabbit kidney, in contrast to apomorphine, which was without effect.7 It is suggested that piribedil has a complex effect on sympathetic transmission. This drug exhibits an ;amphetamine-like' action, causing noradrenaline release from its postganglionic stores. This releasing effect masks an action on prejunctional inhibitory dopamine receptors. In addition, at high doses, piribedil exhibits a marked action on postjunctional sites, since it reduces the vasoconstrictor effect of exogenous noradrenaline.

Hydralazine: effect on the outflow of noradrenaline and mechanical responses evoked by sympathetic nerve stimulation of the rat tail artery.

1 The effects of hydralazine on the vasoconstrictor responses to field stimulation of sympathetic nerves were studied in the isolated proximal segments of the rat tail artery. Vasoconstrictor responses to transmural stimulation were depressed by superfusion of hydrazine (0.3, 3 and 30 muM) in a concentration-dependent manner. The inhibition appeared slowly and was not easily reversed by washing. 2 Hydralazine (30 nM, 0.3 and 3 muM) reduced the stimulation-induced overflow of tritium from proximal and distal segments of the tail artery labelled with [3H]-noradrenaline in a concentration-dependent manner. This phenomenon appeared rapidly and was easily reversed by washing. 3 Theophylline (0.5 mM) did not affect the inhibitory effect of hydralazine on the stimulation-induced tritium efflux from the distal segment of the rat tail artery. 4 The present results indicate that hydralazine has, in addition to its action on vascular smooth muscle, a very marked effect on sympathetic nerve terminals. The mechanism of this presynaptic inhibition appears to be different from the postsynaptic effect, in view of the much shorter delay, the shape of the dose-effect curve, and the lack of interaction with theophylline.

Reduction in hepatic but not in renal and vascular vasopressin receptor number in hyperinsulinemic mice and rats.

Hepatic plasma membranes of female obese mice C57 BL-6 orl ob/ob (ob/ob mice) completely lack vasopressin (VP) receptors of the V1 type whereas kidney VP receptors are normally expressed and functionally coupled to adenylate cyclase. To discover if these alterations are linked to a genetic defect of the V1 receptor, we have studied the binding of VP on liver and kidney membranes of two other models, female diabetic mice C57 BL-6 orl db/db (db/db mice) and female Zucker rats Fatty/orl fa/fa (fa/fa rats), which exhibit different temporal pattern of obesity, hyperinsulinemia and insulin resistance. In addition, since VP is known to exert its vascular response through stimulation of V1 receptors, we have studied the reactivity of VP of isolated tail artery in the three different models, ob/ob and db/db mice and fa/fa rats, and in their respective controls. In all cases, VP kidney receptors and VP vascular reactivity are normal. db/db mice exhibit a marked decrease in hepatic VP receptors whereas a 50% decrease was observed in 32 week fa/fa rats. Angiotensin II and prazosin binding sites are still present as well as the adenylate cyclase response to glucagon. These results suggest that the specific alteration in liver VP receptors is not related to a defect in V1 receptor genetic expression but is specific for liver and appears to parallel the level of hyperinsulinemia and/or insulin resistance.

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.

Mechanism of the depressor response to dopamine in the rat treated with phenoxybenzamine.

Alterations of hypotensive responses to dopamine by antagonists were characterized in alpha-blocked, anaesthetized rats. Responses were not affected by d-propranolol (0.1 mg/kg) whereas d,1-propranolol (0.1 mg/kg) or haloperidol (1.0 mg/kg) attenuated them; higher doses of inhibitors (1.0 mg/kg; 5.0 mg/kg, respectively) failed to produce a higher inhibition, but combinations of low doses abolished the depressor responses. In adrenalectomized rats, hypotensive responses decreased; haloperidol always attenuated the responses while d,1-propranolol became ineffective. Dopamine produced an enhancement of plasma adrenaline and noradrenaline levels, which was decreased by d,1-propranolol and increased by haloperidol. The data suggest that in rats the depressor component of dopamine is due to activation of both dopaminergic and beta-adrenoceptors. The beta component appears to be due to the release of adrenaline. The results also support the concept of the existence in sympathetic nerve endings and adrenal glands of stimulatory beta-adrenergic and inhibitory dopaminergic prejunctional receptors.

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.

Vasopressin inhibition of human platelet adenylate cyclase: variable responsiveness between donors and involvement of a G-protein different from Gi.

There is controversy concerning the inhibitory effect of arginine-vasopressin (AVP) on human platelet adenylate cyclase activity, which putatively involves Gi as the G-protein. To clarify this point, the effects of AVP on human platelet membranes were studied by measuring the activities of the high-affinity GTPase, as an index of G-protein involvement, and of adenylate cyclase. AVP stimulated GTPase activity in a dose-dependent fashion (KAct = 1.1 +/- 0.2 nM) and caused a parallel adenylate cyclase inhibition (KAct = 1.3 +/- 0.7 nM). The extent of these AVP-induced responses varied considerably from one subject to another but they were linearly related, suggesting a causal relationship between the two activities. Moreover, a difference in responsiveness to the inhibitory effects to epinephrine on adenylate cyclase was also observed between donors. Since the AVP- and epinephrine-stimulated GTPase activities were additive at their respective maximal effect, and in view of the lack of linear relationship between AVP- and epinephrine-induced adenylate cyclase inhibition, our results suggest, that in spite of the AVP inhibitory action on platelet adenylate cyclase, the G-protein involved in this effect is different from Gi.

Differential effects of oral trandolapril and enalapril on rat tissue angiotensin-converting enzyme.

Trandolapril (3-100 micrograms/kg) and enalapril (10-300 micrograms/kg) were administered orally to conscious rats. Angiotensin-converting enzyme (CE) activity was inhibited in serum, heart ventricle, renal inner cortex, lung, aorta, adrenal cortex and adrenal medulla, but not in the striatum. Inhibition was maximal at 2 h and with trandolapril was maintained for 24 h. Blood pressure and heart rate were not affected by either compound. Trandolapril was 6-10-fold more potent than enalapril. Differences between trandolapril and enalapril in CE inhibition observed in heart ventricle, adrenal cortex and medulla could be due to the presence of more than one type of CE or CE-like activity.

Influence of Ca2+e on 5-HT2- and alpha 1-induced arterial contraction and phosphoinositide metabolism.

Serotonin and phenylephrine were found to induce contractile responses and inositol phosphate (IP) formation in isolated rat tail artery. Both processes displayed similar concentration dependence in the presence of 2.5 mM external calcium (Ca2+e) and both were respectively inhibited by either ketanserine or prazosin, depending on the agonist used. In the absence of Ca2+e the amines no longer produced a contractile effect. In addition, lack of Ca2+ caused a shift to the right in the dose-response curve for phenylephrine-induced IP formation whereas serotonin-induced IP formation was not affected by changes in Ca2+e. The results suggest that alpha 1- and 5-HT2-induced contractions are quantitatively related to phosphatidylinositol metabolism. Contraction, but not IP formation requires the presence of Ca2+e. Different effects of Ca2+e on phenylephrine- and serotonin-induced IP formation could be related to a differential Ca2+ effect on binding of alpha 1- or 5-HT2 agonists.

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.

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.

V1a-vasopressin specific receptors on human platelets: potentiation by ADP and epinephrine and evidence for homologous down-regulation.

Using very specific vasopressin (VP) analogues, the human platelet VP receptor was characterized as a V1a rather than a V1b receptor, on the basis of the effect of the analogues on shape-change and aggregation. The platelet VP binding sites appeared to be subject to homologous down-regulation by plasma VP, in view of the inverse correlation found between the maximal capacity of binding of tritiated VP to platelets and the immunoreactive VP concentration in poor platelet plasma from the same individual. Aggregating effect of VP on human platelets was potentiated by both ADP and epinephrine. In addition, VP was able to release serotonin from human platelets, but only at high concentration.

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.

Properties of vasopressin-activated human platelet high affinity GTPase.

Effect of 8-arginine vasopressin (AVP) was examined on human platelet membrane GTPase activity as an index of a G-protein involvement. AVP stimulated a high-affinity GTPase activity in a dose-dependent manner (Kact = 1.1 +/- 0.2 nM). This stimulation was blocked by a V1a antagonist, thus confirming the V1a nature of the platelet AVP receptor. There were important variations among individuals in the AVP-induced stimulation of GTPase activity, that were in relation with the AVP-maximal binding capacity. These data suggest a causal relationship between the binding of AVP to its receptor and transduction elicited by a G-protein, without amplification. In addition, in view of the variable AVP responsiveness observed among individuals, platelet AVP-receptor appears to be subject to regulation.