Fluorescent ligands for the study of receptors

McGrath, J.C.; Arribas, Silvia M.; Daly, Craig

doi:10.1016/s0165-6147(96)40004-9

Cited by 96 publications

(103 citation statements)

References 17 publications

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“…Previously, LSCM has been applied in combination with fluorescent ligands (23,24) or antibodies (25) to determine the location of several receptors in blood vessels. This was the approach chosen in this work to study the location and distribution of individual adenosine receptor subtypes (using specific antibodies against each adenosine receptor subtype) in the mesenteric artery adventitia of both SHR and WKY.…”

Section: Presence and Distribution Of Adenosine Receptor Subtypes In mentioning

confidence: 99%

Impaired Inhibitory Function of Presynaptic A1-Adenosine Receptors in SHR Mesenteric Arteries

et al. 2013

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Abstract. In hypertension, vascular reactivity alterations have been attributed to numerous factors, including higher sympathetic innervation/adenosine. This study examined the modulation of adenosine receptors on vascular sympathetic nerves and their putative contribution to higher noradrenaline spillover in hypertension. We assessed adenosine receptors distribution in the adventitia through confocal microscopy, histomorphometry, and their regulatory function on electrically-evoked [3 H]-noradrenaline overflow, using selective agonists/antagonists. We found that: i) A 1 -adenosine receptor agonist (CPA: 100 nM) inhibited tritium overflow to a lower extent in SHR (25% ± 3%, n = 14) compared to WKY (38% ± 3%, n = 14) mesenteric arteries; ii) A 2A -adenosine receptor agonist (CGS 21680: 100 nM) induced a slight increase of tritium overflow that was similar in SHR (22% ± 8%, n = 8) and WKY (24% ± 5%, n = 8) mesenteric arteries; iii) A 2B -and A 3 -adenosine receptors did not alter tritium overflow in either strain; iv) all adenosine receptors were present on mesenteric artery sympathetic nerves and/or some adventitial cells of both strains; and v) A 1 -adenosine receptor staining fractional area was lower in SHR than in WKY mesenteric arteries. We conclude that there is an impaired inhibitory function of vascular presynaptic A 1 -adenosine receptors in SHR, likely related to a reduced presence of these receptors on sympathetic innervation, which might lead to higher levels of noradrenaline in the synaptic cleft and contribute to hypertension in this strain.

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Section: Presence and Distribution Of Adenosine Receptor Subtypes In mentioning

confidence: 99%

Impaired Inhibitory Function of Presynaptic A1-Adenosine Receptors in SHR Mesenteric Arteries

et al. 2013

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“…We used the fluorescent ligand QAPB, an analog of prazosin with high affinity for ␣ 1 -adrenoceptors (pK i ϭ 8.1-8.9) (McGrath et al, 1996;Daly et al, 1998;Mackenzie et al, 2000) but that also has moderate affinity for ␣ 2 -adrenoceptors (pK i ϭ 7.3-7.8; Dr. C. M. Milligan, personal communication; method of Brown et al, 1993). Binding to the three ␣ 1 -adrenoceptors was eliminated by using vessels from the ␣ 1B -adrenoceptor-knockout mouse and blocking the other two subtypes with selective antagonists: for ␣ 1A -adrenoceptors, 5-methylurapidil, (5MeU); and for ␣ 1D -adrenoceptors, BMY7378.…”

Section: Visualization Of Endothelial ␣ 2 -Adrenoceptorsmentioning

confidence: 99%

Two “Knockout” Mouse Models Demonstrate That Aortic Vasodilatation Is Mediated via α_2A-Adrenoceptors Located on the Endothelium

Shafaroudi¹,

McBride²,

Deighan³

et al. 2005

J Pharmacol Exp Ther

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UK-14,304 [5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine]-mediated vasodilator responses were studied on wire myograph-mounted mouse aorta to determine the cells involved, mechanisms of action, and subtypes of ␣ 2 -adrenoceptors. In the presence of induced tone, UK-14,304 produced concentration-related vasodilatation that was abolished by rauwolscine, N -nitro-L-arginine methyl ester (L-NAME), or endothelium removal, indicating that endothelial ␣ 2 -adrenoceptors can release nitric oxide. In the ␣ 2A -adrenoceptor knockout mouse and the D79N mouse, a functional knockout of the ␣ 2A -adrenoceptor, these relaxant effects of UK-14,304 were lost, indicating the involvement of the ␣ 2A -adrenoceptor. UK-14,304 could also contract aorta: a small contraction occurred at high concentrations, was enhanced by L-NAME, and was absent in the ␣ 1D -adrenoceptor knockout mouse, indicating activation of the ␣ 1D -adrenoceptor. There was no evidence for a contractile ␣ 2 -adrenoceptor-mediated response. A fluorescent ligand, quinazoline piperazine bodipy, antagonized the relaxant action of UK-14,304. This compound could be visualized on aortic endothelial cells, and its binding could be prevented by rauwolscine, providing direct evidence for the presence of ␣ 2 -adrenoceptors on the endothelium. Norepinephrine reduced tone in the ␣ 1D -adrenoceptor knockout and controls, an effect blocked by rauwolscine and L-NAME but not by prazosin. This suggests that norepinephrine activates endothelial ␣ 2 -adrenoceptors. In conclusion, the endothelium of mouse aorta has an ␣ 2A -adrenoceptor that responds to norepinephrine; promotes the release of nitric oxide, causing smooth muscle relaxation; and that can be directly visualized. Knockout or genetic malfunction of this receptor should increase arterial stiffness, exacerbated by raised catecholamines, and contribute to heart failure.All three ␣ 2 -adrenoceptors have distinct, yet poorly defined, roles in the control of the vascular system. The limited selectivity of agonists and antagonists has therefore prompted the use of transgenic mouse models. The subtypes are ␣ 2A , ␣ 2B , and ␣ 2C : the mouse ortholog of the human ␣ 2A -adrenoceptor is sometimes called the ␣ 2D -or ␣ 2A/D -adrenoceptor; we use the generic term ␣ 2A -adrenoceptor (Alexander et al., 2004). They have two direct pharmacological effects on blood vessels that can modify vascular tone: a direct vasopressor action (for review, see Wilson et al., 1991;Guimaraes and Moura, 2001) and vasodilatation via endothelium-derived relaxant factors (Cocks and Angus, 1983;Vanhoutte, 2001). They also reduce sympathetic traffic centrally and inhibit transmitter release from sympathetic postganglionic nerves (Starke, 2001), although this is not well established as a physiological phenomenon in blood vessels. Available pharmacological data and knockout studies, although not definitive, present evidence for, at least, ␣ 2A -, ␣ 2B -, and ␣ 2C -adrenoceptors for vasoconstriction, ␣ 2A -and ␣ 2C -adrenoceptors for sympatho-...

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“…This allows quantitative visual assessment of its binding to receptors at equilibrium . It has been used to measure binding affinity at recombinant and native ␣ 1 -ARs in different regions of cultured cells and of freshly dissociated cells from human and other mammalian tissue (McGrath et al, 1996(McGrath et al, , 1999Daly et al, 1998;Mackenzie et al, 2000). We have quantified the pharmacology of agonist and antagonist interaction at recombinant ␣ 1 -AR in R-1Fs , demonstrating that the receptors are functional and have the predicted pharmacology.…”

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confidence: 99%

β-Arrestin-Dependent Spontaneous α_1a-Adrenoceptor Endocytosis Causes Intracellular Transportation of α-Blockers via Recycling Compartments

Pediani¹,

Colston²,

Caldwell³

et al. 2004

Mol Pharmacol

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The antagonist ligand BODIPY-FL-prazosin (QAPB) fluoresces when bound to bovine ␣ 1a -adrenoceptors (ARs). Data indicate that the receptor-ligand complex is spontaneously internalized by ␤-arrestin-dependent endocytosis. Internalization of the ligand did not occur in ␤-arrestin-deficient cells; was blocked or reversed by another ␣ 1 ligand, phentolamine, indicating it to reflect binding to the orthosteric recognition site; and was prevented by blocking clathrin-mediated endocytosis. The ligand showed rapid, diffuse, low-intensity, surface binding, superseded by punctate intracellular binding that developed to equilibrium in 50 to 60 min and was reversible on ligand removal, indicating a dynamic equilibrium. In cells expressing a human ␣ 1a -AR-enhanced green fluorescent protein (EGFP) 2 fusion protein, BODIPY-R-558/568-prazosin (RQAPB) colocalized with the fusion, indicating that the ligand gained access to all compartments containing the receptor, and, conversely, that the receptor has affinity for the ligand at all of these sites. The distribution of QAPB binding sites was similar for receptors with or without EGFP2, validating the fusion protein as an indicator of receptor location. The ligand partially colocalized with ␤-arrestin in recycling and late endosomes, indicating receptor transit without destruction. Organelles containing receptors showed considerable movement consistent with a transportation function. This was absent in ␤-arrestin-deficient cells, indicating that both constitutive receptor internalization and subsequent intracellular transportation are ␤-arrestin-dependent. Calculations of relative receptor number indicate that at steady state, less than 30% of receptors reside on the cell surface and that recycling is rapid. We conclude that ␣ 1a -ARs recycle rapidly by an agonist-independent, constitutive, ␤-arrestin-dependent process and that this can transport "␣-blockers" into cells carrying these receptors.

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Fluorescent ligands for the study of receptors

Cited by 96 publications

References 17 publications

Impaired Inhibitory Function of Presynaptic A1-Adenosine Receptors in SHR Mesenteric Arteries

Impaired Inhibitory Function of Presynaptic A1-Adenosine Receptors in SHR Mesenteric Arteries

Two “Knockout” Mouse Models Demonstrate That Aortic Vasodilatation Is Mediated via α_2A-Adrenoceptors Located on the Endothelium

β-Arrestin-Dependent Spontaneous α_1a-Adrenoceptor Endocytosis Causes Intracellular Transportation of α-Blockers via Recycling Compartments

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Fluorescent ligands for the study of receptors

Cited by 96 publications

References 17 publications

Impaired Inhibitory Function of Presynaptic A1-Adenosine Receptors in SHR Mesenteric Arteries

Impaired Inhibitory Function of Presynaptic A1-Adenosine Receptors in SHR Mesenteric Arteries

Two “Knockout” Mouse Models Demonstrate That Aortic Vasodilatation Is Mediated via α2A-Adrenoceptors Located on the Endothelium

β-Arrestin-Dependent Spontaneous α1a-Adrenoceptor Endocytosis Causes Intracellular Transportation of α-Blockers via Recycling Compartments

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Product

Resources

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Two “Knockout” Mouse Models Demonstrate That Aortic Vasodilatation Is Mediated via α_2A-Adrenoceptors Located on the Endothelium

β-Arrestin-Dependent Spontaneous α_1a-Adrenoceptor Endocytosis Causes Intracellular Transportation of α-Blockers via Recycling Compartments