Fluorescent localization of the beta-adrenergic receptor on DDT-1 cells. Down-regulation by adrenergic agonists.
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ABSTRACT:
SUBMITTER: Zemcik BA
PROVIDER: S-EPMC1149007 | biostudies-other | 1988 Apr
REPOSITORIES: biostudies-other
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Project description:Astrocytomas and glioblastomas have been particularly difficult to treat and refractory to chemotherapy. However, significant evidence has been presented that demonstrates a decrease in astrocytoma cell proliferation subsequent to an increase in cAMP levels. The 1321N1 astrocytoma cell line, as well as other astrocytomas and glioblastomas, expresses ?(2)-adrenergic receptors (?(2)-ARs) that are coupled to G(s) activation and consequent cAMP production. Experiments were conducted to determine whether the ?(2)-AR agonist (R,R')-fenoterol and other ?(2)-AR agonists could attenuate mitogenesis and, if so, by what mechanism. Receptor binding studies were conducted to characterize ?(2)-AR found in 1321N1 and U118 cell membranes. In addition, cells were incubated with (R,R')-fenoterol and analogs to determine their ability to stimulate intracellular cAMP accumulation and inhibit [(3)H]thymidine incorporation into the cells. 1321N1 cells contain significant levels of ?(2)-AR as determined by receptor binding. (R,R')-fenoterol and other ?(2)-AR agonists, as well as forskolin, stimulated cAMP accumulation in a dose-dependent manner. Accumulation of cAMP induced a decrease in [(3)H]thymidine incorporation. There was a correlation between concentration required to stimulate cAMP accumulation and inhibit [(3)H]thymidine incorporation. U118 cells have a reduced number of ?(2)-ARs and a concomitant reduction in the ability of ?(2)-AR agonists to inhibit cell proliferation. These studies demonstrate the efficacy of ?(2)-AR agonists for inhibition of growth of the astrocytoma cell lines. Because a significant portion of brain tumors contain ?(2)-ARs to a greater extent than whole brain, (R,R')-fenoterol, or some analog, may be useful in the treatment of brain tumors after biopsy to determine ?(2)-AR expression.
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Project description:Trace amine-associated receptors (TAAR) are rhodopsin-like G-protein-coupled receptors (GPCR). TAAR are involved in modulation of neuronal, cardiac and vascular functions and they are potentially linked with neurological disorders like schizophrenia and Parkinson's disease. Subtype TAAR1, the best characterized TAAR so far, is promiscuous for a wide set of ligands and is activated by trace amines tyramine (TYR), phenylethylamine (PEA), octopamine (OA), but also by thyronamines, dopamine, and psycho-active drugs. Unfortunately, effects of trace amines on signaling of the two homologous β-adrenergic receptors 1 (ADRB1) and 2 (ADRB2) have not been clarified yet in detail. We, therefore, tested TAAR1 agonists TYR, PEA and OA regarding their effects on ADRB1/2 signaling by co-stimulation studies. Surprisingly, trace amines TYR and PEA are partial allosteric antagonists at ADRB1/2, whereas OA is a partial orthosteric ADRB2-antagonist and ADRB1-agonist. To specify molecular reasons for TAAR1 ligand promiscuity and for observed differences in signaling effects on particular aminergic receptors we compared TAAR, tyramine (TAR) octopamine (OAR), ADRB1/2 and dopamine receptors at the structural level. We found especially for TAAR1 that the remarkable ligand promiscuity is likely based on high amino acid similarity in the ligand-binding region compared with further aminergic receptors. On the other hand few TAAR specific properties in the ligand-binding site might determine differences in ligand-induced effects compared to ADRB1/2. Taken together, this study points to molecular details of TAAR1-ligand promiscuity and identified specific trace amines as allosteric or orthosteric ligands of particular β-adrenergic receptor subtypes.
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Project description:A key question in receptor signaling is how specificity is realized, particularly when different receptors trigger the same biochemical pathway(s). A notable case is the two β-adrenergic receptor (β-AR) subtypes, β1 and β2, in cardiomyocytes. They are both coupled to stimulatory Gs proteins, mediate an increase in cyclic adenosine monophosphate (cAMP), and stimulate cardiac contractility; however, other effects, such as changes in gene transcription leading to cardiac hypertrophy, are prominent only for β1-AR but not for β2-AR. Here, we employ highly sensitive fluorescence spectroscopy approaches, in combination with a fluorescent β-AR antagonist, to determine the presence and dynamics of the endogenous receptors on the outer plasma membrane as well as on the T-tubular network of intact adult cardiomyocytes. These techniques allow us to visualize that the β2-AR is confined to and diffuses within the T-tubular network, as opposed to the β1-AR, which is found to diffuse both on the outer plasma membrane as well as on the T-tubules. Upon overexpression of the β2-AR, this compartmentalization is lost, and the receptors are also seen on the cell surface. Such receptor segregation depends on the development of the T-tubular network in adult cardiomyocytes since both the cardiomyoblast cell line H9c2 and the cardiomyocyte-differentiated human-induced pluripotent stem cells express the β2-AR on the outer plasma membrane. These data support the notion that specific cell surface targeting of receptor subtypes can be the basis for distinct signaling and functional effects.
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Project description:The β3 -adrenergic receptor (β3 -AR) is found in several tissues such as adipose tissue and urinary bladder. It is a therapeutic target because it plays a role in thermogenesis, lipolysis, and bladder relaxation. Two β3 -AR agonists are used clinically: mirabegron 1 and vibegron 2, which are indicated for overactive bladder syndrome. However, these drugs show adverse effects, including increased blood pressure in mirabegron patients. Hence, new β3 -AR agonists are needed as starting points for drug development. Previous pharmacophore modeling studies of the β3 -AR did not involve experimental in vitro validation. Therefore, this study aimed to conduct prospective virtual screening and confirm the biological activity of virtual hits. Ligand-based pharmacophore modeling was performed since no 3D structure of human β3 -AR is yet available. A dataset consisting of β3 -AR agonists was prepared to build and validate the pharmacophore models. The best model was employed for prospective virtual screening, followed by physicochemical property filtering and a docking evaluation. To confirm the activity of the virtual hits, an in vitro assay was conducted, measuring cAMP levels at the cloned β3 -AR. Out of 35 tested compounds, 4 compounds were active in CHO-K1 cells expressing the human β3 -AR, and 8 compounds were active in CHO-K1 cells expressing the mouse β3 -AR.
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Project description:Chinese hamster ovary cell constructs expressing either the β 1-, β 2- or β 3-adrenergic receptor (AR) were used to determine whether a novel β-AR modulator, lubabegron fumarate (LUB; Experior, Elanco Animal Health) might exert greater potency for a specific β-AR subtype. EC50 values calculated based on cAMP accumulation in dose response curves indicate that LUB is highly selective for the β 3-AR subtype, with an EC50 of 6 × 10-9 M, with no detectible agonistic activity at the β 2-AR. We hypothesized that the accumulation of lipolytic markers would reflect the agonist activity at each of the β-receptor subtypes of the specific ligand; additionally, there would be differences in receptor subtype expression in subcutaneous (s.c.) and intrmuscular (i.m.) adipose tissues. Total RNA was extracted from adipose tissue samples and relative mRNA levels for β 1-, β2-, and β 3-AR were measured using real-time quantitative polymerase chain reaction. Fresh s.c. and i.m. adipose tissue explants were incubated with isoproterenol hydrochloride (ISO; β-AR pan-agonist), dobutamine hydrochloride (DOB; specific β 1-AA), salbutamol sulfate (SAL; specific β 2-AA), ractopamine hydrochloride (RAC), zilpaterol hydrochloride (ZIL), BRL-37344 (specific β 3-agonist), or LUB for 30 min following preincubation with theophylline (inhibitor of phosphodiesterase). Relative mRNA amounts for β 1-, β 2-, and β 3-AR were greater (P < 0.05) in s.c. than in i.m. adipose tissue. The most abundant β-AR mRNA in both adipose tissues was the β 2-AR (P < 0.05), with the β 1- and β 3-AR subtypes being minimally expressed in i.m. adipose tissue. ISO, RH, and ZH stimulated the release of glycerol and nonesterified fatty acid (NEFA) from s.c. adipose tissue, but these β-AR ligands did not alter concentrations of these lipolytic markers in i.m. adipose tissue. LUB did not affect glycerol or NEFA concentrations in s.c. or i.m. adipose tissue, but attenuated (P < 0.05) the accumulation of cAMP mediated by the β 1- and β 2-AR ligands DOB and SAL in s.c. adipose tissue. Collectively, these data indicate that bovine i.m. adipose tissue is less responsive than s.c. adipose tissue to β-adrenergic ligands, especially those that are agonists at the β 1- and β3-receptor subtypes. The minimal mRNA expression of the β 1- and β 3 subtypes in i.m. adipose tissue likely limits the response potential to agonists for these β-AR subtypes.
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