P719Endothelial expression of adenylate cyclase type 9 (Adcy9) regulates endothelial cell signalling and vasodilation

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
Y Rautureau ◽  
M Berlatie ◽  
D Rivas ◽  
K Uy ◽  
G Miquel ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Adenylate Cyclase ◽  
Ex Vivo ◽  
Primary Cultures ◽  
Adenosine Monophosphate ◽  
Clinical Effects ◽  
Camp Accumulation ◽  
Human Coronary Artery ◽  
Femoral Arteries ◽  
Mechanical Removal

Abstract Background/Introduction The clinical benefits of the cholesterol ester transfer protein (CETP) inhibitor dalcetrapib are dependent on the ADCY9 gene, which encodes adenylate cyclase (AC) type 9 (AC9). AC9 is one of nine membrane-bound isoforms of AC producing cyclic adenosine monophosphate (cAMP). We demonstrated that Adcy9 inactivation in the mouse protects from atherosclerosis, but only in absence of CETP. Adcy9 inactivation is also associated with improved endothelial function, including greater endothelial-dependent vasodilation (EDV) in response to acetylcholine (ACh). This suggests that Adcy9 may control endothelial Ca2+, an essential mediator of endothelial cell (EC) signalling. We hypothesized that Adcy9 is expressed in the endothelium and controls EC signalling. Purpose Our aim was to study AC9 expression in the vascular endothelium and the role of AC9 expression on endothelial cAMP signalling and Ca2+ dynamics. Methods The effects of Adcy9 inactivation were studied using wild-type (WT) and Adcy9-inactivated (Adcy9Gt/Gt) mice and siRNA-mediated inactivation of ADCY9 in human coronary artery endothelial cells (HCAEC). In the mouse, AC9 expression was quantified in whole aorta (± endothelium) and primary cultures of lung EC (LEC) by Western blot. Endothelial Ca2+ pulsars were monitored in mouse femoral arteries in response to acetylcholine (ACh, 10 μM). cAMP accumulation to AC activators VIP and forskolin were measured in LEC and HCAEC. EDV to VIP was studied ex vivo in mouse femoral arteries using pressurized arteriography. Results AC9 is expressed in LEC and the aorta, and its detection in the latter decreased by 33% after mechanical removal of the endothelium (P<0.05). AC activation with forskolin (10–4 M) in LEC led to increased cAMP accumulation in response to Adcy9 inactivation (WT: 1129±138 pmol cAMP/mg of protein, Adcy9Gt/Gt: 1965±169, n=3; P<0.01). ADCY9 inactivation in HCAEC also increased cAMP accumulation to forskolin 10–4M (control siRNA: 85±17, ADCY9 siRNA: 150±6, n=3; P<0.01). In LEC, receptor-dependent accumulation of cAMP to VIP (10–5 M) was higher in Adcy9Gt/Gt (1509±258)) compared to WT (915±170, n=3; P<0.05) mice. In mouse femoral arteries, VIP-induced maximal vasodilation was increased by Adcy9 inactivation in the presence of the endothelium (Adcy9Gt/Gt: 85±4%, n=6; WT: 52±9%, n=5, P<0.05), but not in its absence (Adcy9Gt/Gt: 67±12%, n=6 and WT: 59±15%, n=5). In the femoral artery endothelium, ACh increased Ca2+ pulsar frequency more in Adcy9Gt/Gt (243±32%, n=4) than in WT (178±19%, n=5; P<0.05) mice. Conclusion ADCY9 is expressed in the endothelium, and its inactivation potentiates endothelial cell Ca2+ dynamics, cAMP accumulation and VIP-induced vasodilation. We therefore identify ADCY9 as a new molecular pathway regulating endothelial-dependent vasodilation. This suggests that ADCY9's endothelial biology could be involved in the ADCY9 genotype-dependent clinical effects of dalcetrapib. Acknowledgement/Funding DalCor

Plasticity of endothelial cells: rapid dedifferentiation of freshly isolated high endothelial venule endothelial cells outside the lymphoid tissue microenvironment

Blood ◽  
2004 ◽  
Vol 103 (11) ◽  
pp. 4164-4172 ◽  
Author(s):  
Delphine-Armelle Lacorre ◽  
Espen S. Baekkevold ◽  
Ignacio Garrido ◽  
Per Brandtzaeg ◽  
Guttorm Haraldsen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Lymphoid Tissue ◽  
Ex Vivo ◽  
Primary Cultures ◽  
High Endothelial Venule ◽  
Tissue Specific ◽  
Tissue Microenvironment ◽  
Cell Phenotypes

Abstract Endothelial cells display remarkable heterogeneity in different organs and vascular beds. Although many studies suggest that tissues “speak” to endothelial cells, endothelial cell diversity remains poorly characterized at the molecular level. Here, we describe a novel strategy to characterize tissue-specific endothelial cell phenotypes and to identify endothelial cell genes that are under the control of the local microenvironment. By comparing post-capillary high endothelial venule endothelial cells (HEVECs), freshly isolated from human tonsils without any cell culture step, with HEVECs cultured for 2 days, we found that HEVECs rapidly lost their specialized characteristics when isolated from the lymphoid tissue microenvironment. Striking changes occurred as early as after 48 hours, with complete loss of the postcapillary venule–specific Duffy antigen receptor for chemokines (DARCs) and the HEV-specific fucosyltransferase Fuc-TVII. DNA microarray analysis identified several other candidate HEV genes that were rapidly down-regulated ex vivo, including type XV collagen, which we characterized as a novel, abundant HEV transcript in situ. Together, our results demonstrate that blood vessel type–specific and tissue-specific characteristics of endothelial cells are under the control of their microenvironment. Therefore, even short-term primary cultures of human endothelial cells may not adequately mimic the differentiated endothelial cell phenotypes existing in vivo.

Effect of P1-purinergic agonist on thyrotropin stimulation of H2O2 generation in FRTL-5 and porcine thyroid cells

1994 ◽  
Vol 130 (2) ◽  
pp. 180-186 ◽  
Author(s):  
Ulla Björkman ◽  
Ragnar Ekholm
Keyword(s):  
Adenylate Cyclase ◽  
Primary Culture ◽  
Purinergic Receptor ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Camp Accumulation ◽  
High Concentration ◽  
Thyroid Cells ◽  
H2o2 Generation ◽  
Stimulation Of

Björkman U, Ekholm R. Effect of P1-purinergic agonist on thyrotropin stimulation of H2O2 generation in FRTL-5 and porcine thyroid cells. Eur J Endocrinol 1994;130:180–6. ISSN 0804–4643 Our previous studies have shown that the generation of H2O2 in FRTL-5 thyroid cells is regulated via both the adenylate cyclase/cyclic adenosine monophosphate (cAMP) and Ca2+/phosphatidylinositol pathway: thyrotropin (TSH) stimulates H2O2 generation through both pathways, via the former at a low concentration and via the latter at a high concentration. In porcine thyrocytes in primary culture H2O2 generation is stimulated only via the Ca2+/phosphatidylinositol route. In the present study we explored the effect of a P1-purinergic agonist (phenylisopropyladenosine, PIA) on stimulations induced by TSH and by adenosine triphosphate (ATP), an activator of the Ca2+/phosphatidylinositol cascade via the P2-purinergic receptor. In FRTL- 5 cells, PIA potentiated H2O2 generation stimulated by TSH at 10U/l (but not at 1 U/l), Ca2+ mobilization induced by TSH and Ca2+ mobilization induced by ATP at 1 μmol/l (but not 10 μmol/l). Phenylisopropyladenosine strongly inhibited TSH-induced cAMP accumulation in FRTL-5 cells. In pig thyrocytes, PIA had no effect on H2O2 generation stimulated by TSH or ATP and no effect on ATP-stimulated Ca2+ mobilization. Also, PIA did not inhibit TSH-stimulated cAMP accumulation in pig thyrocytes, and by itselfhad no effecton H2O2 generation or Ca2 + mobilization. Thus, in FRTL-5 cells, but not in porcine thyrocytes, PIA modulates TSH-stimulated H2O2 generation by enhancing the Ca2+/phosphatitylinositol route and inhibiting the adenylate cyclase/cAMP route of the TSH signal. The net result of this modulation apparently depends on the balance between inhibition of the cAMP route and enhancement of the Ca2+ route. This may explain the lack of potentiation observed by 1 U/1 TSH. Ragnar Ekholm, Department of Anatomy, Medicinaregatan 3, S-413 90 Göteborg, Sweden

scholarly journals Nanostructured Lipid Carriers Deliver Resveratrol, Restoring Attenuated Dilation in Small Coronary Arteries, via the AMPK Pathway

Biomedicines ◽  
2021 ◽  
Vol 9 (12) ◽  
pp. 1852
Author(s):  
Cai Astley ◽  
Chahinez Houacine ◽  
Azziza Zaabalawi ◽  
Fiona Wilkinson ◽  
Adam P. Lightfoot ◽  
...  
Keyword(s):  
Coronary Arteries ◽  
Antihypertensive Drugs ◽  
Ex Vivo ◽  
Adenosine Monophosphate ◽  
Nanostructured Lipid Carriers ◽  
Human Coronary Artery ◽  
Ex Vivo Model ◽  
Sustained Drug Release ◽  
Acute Hypertension ◽  
Pressure Elevation

Nanostructured lipid carriers (NLCs) are an emerging drug delivery platform for improved drug stability and the bioavailability of antihypertensive drugs and vasoprotective nutraceutical compounds, such as resveratrol (RV). The objective of this study was to ascertain NLCs’ potential to deliver RV and restore attenuated dilator function, using an ex vivo model of acute hypertension. Trimyristin–triolein NLCs were synthesized and loaded with RV. The uptake of RV-NLCs by human coronary artery endothelial cells (HCAECs) maintained their viability and reduced both mitochondrial and cytosolic superoxide levels. Acute pressure elevation in isolated coronary arteries significantly attenuated endothelial-dependent dilator responses, which were reversed following incubation in RV-NLCs, superoxide dismutase or apocynin (p < 0.0001). RV-NLCs demonstrated a five-fold increase in potency in comparison to RV solution. At elevated pressure, in the presence of RV-NLCs, incubation with Nω-nitro-l-arginine (L-NNA) or indomethacin resulted in a significant reduction in the restored dilator component (p < 0.0001), whereas apamin and TRAM-34 had no overall effect. Incubation with the adenosine monophosphate-activated protein kinase (AMPK) inhibitor dorsomorphin significantly attenuated dilator responses (p < 0.001), whereas the SIRT-1 inhibitor EX-527 had no effect. RV-NLCs improved the impaired endothelial-dependent dilation of small coronary arteries, following acute pressure elevation, via NO and downstream COX elements, mediated by AMPK. We suggest that RV-NLCs are an effective delivery modality for improved potency and sustained drug release into the vasculature. Our findings have important implications for the future design and implementation of antihypertensive treatment strategies.

Pentobarbital Enhances Cyclic Adenosine Monophosphate Production in the Brain by Effects on Neurons but Not Glia

1996 ◽  
Vol 84 (5) ◽  
pp. 1148-1155 ◽  
Author(s):  
Jerry M. Gonzales ◽  
Iris Mendez-Bobe
Keyword(s):  
Cerebral Cortex ◽  
Cyclic Amp ◽  
Cortical Neurons ◽  
Neuronal Excitability ◽  
Primary Cultures ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Camp Accumulation ◽  
Camp Production ◽  
The Brain

Background Cyclic adenosine monophosphate (cAMP) is an important regulator of neuronal excitability. The effects of barbiturates on cAMP production in intact neurons are not known. This study used cultures of cortical neurons, cultures of glia, and slices of cerebral cortex from the rat to study the effects of barbiturates on cAMP regulation in the brain. Methods Primary cultures of cortical neurons or glia were prepared from 17-day gestational Sprague-Dawley rat fetuses and were used after 12-16 days in culture. Cross-cut slices (300 microns) were prepared from cerebral cortex of adult rats. Cyclic AMP accumulation was determined by measuring the conversion of [3H]adenosine triphosphate (ATP) to [3H]cAMP in cells preloaded with [3H]adenine. Results Pentobarbital enhanced isoproterenol- and forskolin-stimulated, but not basal, cAMP accumulation in cultures of cerebral neurons. Cyclic AMP production was enhanced by pentobarbital in a dose-dependent fashion up to a concentration of 250 microM; This concentration of pentobarbital increased cAMP production by 40-50% relative to that in controls without pentobarbital. At 500 microM pentobarbital, the magnitude of the enhancement was less. Pentobarbital had no effect on isoproterenol-stimulated cAMP production in cultures containing only glia. Pentobarbital also enhanced isoproterenol-stimulated, but not basal, cAMP production in slices of cerebral cortex by approximately 30% at concentrations of 62.5-250 microM and by almost 100% at 500 microM. Conclusions Pentobarbital enhances stimulated cAMP accumulation in cultured preparations from brain and fresh cortical slices. Neurons are required for this effect. Because cAMP modulates neuronal excitability, this effect of pentobarbital may be an important mechanism by which this anesthetic influences brain function.

Ticlopidine and Clopidogrel (SR 25990C) Selectively Neutralize ADP Inhibition of PGE1-Activated Platelet Adenylate Cyclase in Rats and Rabbits

1991 ◽  
Vol 65 (02) ◽  
pp. 186-190 ◽  
Author(s):  
G Defreyn ◽  
C Gachet ◽  
P Savi ◽  
F Driot ◽  
J P Cazenave ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Adenylate Cyclase ◽  
Direct Effect ◽  
Cyclic Nucleotide ◽  
Camp Accumulation ◽  
Rabbit Platelets ◽  
Camp Levels ◽  
Kinetics Of ◽  
Camp Elevation ◽  
Rat Platelets

SummaryTiclopidine and its potent analogue, clopidogrel, are powerful inhibitors of ADP-induced platelet aggregation. In order to improve the understanding of this ADP-selectivity, we studied the effect of these compounds on PGE1-stimulated adenylate cyclase and on the inhibition of this enzyme by ADP, epinephrine and thrombin. Neither drug changed the basal cAMP levels nor the kinetics of cAMP accumulation upon PGEj-stimulation in rat or rabbit platelets, which excludes any direct effect on adenylate cyclase or on cyclic nucleotide phosphodiesterase. However, the drop in cAMP levels observed after addition of ADP to PGEr stimulated control platelets was inhibited in platelets from treated animals. In contrast, the drop in cAMP levels produced by epinephrine was not prevented by either drug in rabbit platelets. In rat platelets, thrombin inhibited the PGEX-induced cAMP elevation but this effect seems to be entirely mediated by the released ADP. Under these conditions, it was not surprising to find that clopidogrel also potently inhibited that effect of thrombin on platelet adenylate cyclase. In conclusion, ticlopidine and clopidogrel selectively neutralize the ADP inhibition of PGEr activated platelet adenylate cyclase in rats and rabbits.

Organische Nitrate und Thrombozytenfunktion

1988 ◽  
Vol 08 (02) ◽  
pp. 90-99 ◽  
Author(s):  
H. Schröder ◽  
K. Schrör
Keyword(s):  
Endothelial Cell ◽  
Angina Pectoris ◽  
Ex Vivo

ZusammenfassungOrganische Nitrate unterschiedlicher chemischer Struktur sowie Nitroprussidnatrium und Molsidomin (bzw. ihre biologisch aktiven Metaboliten) können die (primäre) Aggregation und Sekretion von Humanthrombozyten in vitro und ex vivo hemmen. Eine solche Wirkung wird für Molsidomin (SIN-1) und Nitroprussidnatrium in vitro in Konzentrationen beobachtet, die in der gleichen Größenordnung liegen wie die vasodilatierenden Effekte der Substanzen. Dagegen sind für eine direkte Antiplättchenwirkung organischer Nitrate (Glyzeryltrinitrat, Isosorbiddinitr at, Isosorbidmononitrate, Teopranitol) in vitro Konzentrationen erforderlich, die ca. 100- bis 1000fach höher sind als die Plasmaspiegel der Substanzen nach therapeutischer Dosierung bzw. die Konzentrationen, die isolierte Gefäßstreifen relaxieren. Als gemeinsamer Wirkungsmechanismus der direkten thrombozy-tenfunktionshemmenden und gefäßerweiternden Wirkung all dieser Substanzen kann heute eine Stickoxid-(NO)-vermittelte Stimulation der cGMP-Bildung angenommen werden, das aus organischen Nitraten als »Pro-drug« entsteht. Die Freisetzung von NO, eines »endothelial cell-derived relaxing factors« (EDRF) aus Nitroprussidnatrium und SIN-1 erfolgt spontan. Dagegen erfordert die Freisetzung von NO aus organischen Nitraten einen enzymatischen Stoffwechselweg, der in isolierten Thrombozyten nicht vorhanden ist. Eine Antiplättchenwirkung organischer Nitrate in vivo bzw. ex vivo wird daher über die Stimulation eines endothelialen, thrombozyteninhibitorischen Faktors erklärt. Hierbei sind Prostazyklin sowie ein bisher unbekannter Endothel-zellfaktor neben einer synergistischen Wirkung organischer Nitrate mit endogenem Prostazyklin in Diskussion. Eine thrombozytenfunktionshemmen-de Wirkung organischer Nitrate könnte in Kombination mit ihren hämody-namischen Effekten auch für die an-tianginöse Wirkung in der Klinik bedeutsam sein, insbesondere zur Verhinderung vasospastischer Zustände bei der instabilen Angina pectoris.

scholarly journals Octreotide and Pasireotide Combination Treatment in Somatotroph Tumor Cells: Predominant Role of SST2 in Mediating Ligand Effects

Cancers ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1816
Author(s):  
Jessica Amarù ◽  
Federica Barbieri ◽  
Marica Arvigo ◽  
Agnese Solari ◽  
Adriana Bajetto ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Tumor Cells ◽  
Intracellular Signaling ◽  
Somatostatin Receptor ◽  
Combined Treatment ◽  
Primary Cultures ◽  
Receptor Subtype ◽  
Camp Accumulation ◽  
Gh Secretion ◽  
Somatostatin Receptor Ligands

First-generation somatostatin receptor ligands (fg-SRLs), such as octreotide (OCT), represent the first-line medical therapy in acromegaly. Fg-SRLs show a preferential binding affinity for somatostatin receptor subtype-2 (SST2), while the second-generation ligand, pasireotide (PAS), has high affinity for multiple SSTs (SST5 > SST2 > SST3 > SST1). Whether PAS acts via SST2 in somatotroph tumors, or through other SSTs (e.g., SST5), is a matter of debate. In this light, the combined treatment OCT+PAS could result in additive/synergistic effects. We evaluated the efficacy of OCT and PAS (alone and in combination) on growth hormone (GH) secretion in primary cultures from human somatotroph tumors, as well as on cell proliferation, intracellular signaling and receptor trafficking in the rat GH4C1 cell line. The results confirmed the superimposable efficacy of OCT and PAS in reducing GH secretion (primary cultures), cell proliferation, cAMP accumulation and intracellular [Ca2+] increase (GH4C1 cells), without any additive effect observed for OCT+PAS. In GH4C1 cells, co-incubation with a SST2-selective antagonist reversed the inhibitory effect of OCT and PAS on cell proliferation and cAMP accumulation, while both compounds resulted in a robust internalization of SST2 (but not SST5). In conclusion, OCT and PAS seem to act mainly through SST2 in somatotroph tumor cells in vitro, without inducing any additive/synergistic effect when tested in combination.

μ-Opiate Receptors in Neuronal Primary Cultures from Cerebral Cortex

1990 ◽  
Vol 17 (3) ◽  
pp. 177-181
Author(s):  
Peter S. Eriksson ◽  
Elisabeth Hansson ◽  
Lars Rönnbäck
Keyword(s):  
Cerebral Cortex ◽  
Primary Cultures ◽  
Opiate Receptors ◽  
Neuronal Cultures ◽  
Camp Accumulation ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Neuronal Primary Cultures ◽  
Dose Dependent ◽  
Μ Receptor

The presence of μ-opioid receptors was demonstrated as effects of receptor stimulation on PGE1-induced cAMP accumulation in neuronal-enriched primary cultures from rat cerebral cortex. Morphine was used as a μ-receptor agonist. There was a dose-dependent inhibition of the PGE1-stimulated cAMP accumulation by morphine, blocked by the μ-receptor antagonist naloxone. These findings suggest that these neuronal cultures express μ-receptors, possibly connected to adenylate cyclase via an inhibitory Gi-protein. The probable use of functional μ-receptors in neurotoxicological tests is discussed.

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