Different Sensitivity of α2A-C10 and α2C-C4 Receptor Subtypes in Coupling to Inhibition of cAMP Accumulation

doi:10.1006/bbrc.1994.1309

Biochemical and Biophysical Research Communications

Volume 199, Issue 2, 15 March 1994, Pages 869-875

https://doi.org/10.1006/bbrc.1994.1309 Get rights and content

Abstract

Two human α₂-adrenergic receptor subtypes, α₂A-C10 and α₂C-C4, were compared with respect to their ability to inhibit stimulated cAMP-production. The inhibition was transduced with about one order of magnitude higher sensitivity in the α₂C-C4 subtype than in the α₂A-C10 subtype. The phorbol ester, TPA, known to desensitize α₂-adrenergic receptor function, possible through phosphorylation of G_i, almost completely abolished the inhibition of cAMP-production in the α₂C-C4 subtype, while only a partial effect was seen in the α₂A-C10 subtype. These results suggest that the receptor subtypes differ with respect to their coupling efficiency to adenylyl cyclase.

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Cited by (16)

Quercetin-3-O-glucuronide inhibits noradrenaline-promoted invasion of MDA-MB-231 human breast cancer cells by blocking β<inf>2</inf>-adrenergic signaling
2014, Archives of Biochemistry and Biophysics
Citation Excerpt :
The intensity of the bands were quantified with a scanner (EPSON, GT-9700F, Japan). Binding assays for β2-AR were carried out as previously described with some modifications [43–45]. Briefly, cells were seeded at a concentration of 10,000–50,000 cells/well in 10 cm dishes in culture media supplemented with 10% FBS, and incubated at 37 °C for 48–72 h. Cells were then washed three times with PBS and treated with propranolol, non-labeled NA and Q3G (from 10−10 to 10−4 M) for 1 h.
Endogenous catecholamines such as adrenaline (A) and noradrenaline (NA) are released from the adrenal gland and sympathetic nervous system during exposure to stress. The adrenergic system plays a central role in stress signaling, and excessive stress was found to be associated with increased production of reactive oxygen species (ROS). Overproduction of ROS induces oxidative damage in tissues and causes the development of diseases such as cancer. In this study, we investigated the effects of quercetin-3-O-glucuronide (Q3G), a circulating metabolite of quercetin, which is a type of natural flavonoid, on the catecholamine-induced β₂-adrenergic receptor (β₂-AR)-mediated response in MDA-MB-231 human breast cancer cells expressing β₂-AR. Treatment with A or NA at concentrations above 1 μM generated significant levels of ROS, and NA treatment induced the gene expression of heme oxygenase-1 (HMOX1), and matrix metalloproteinase-2 (MMP-2) and -9 (MMP9). Inhibitors of p38 MAP kinase (SB203580), cAMP-dependent protein kinase (PKA) (H-89), activator protein-1 (AP-1) transcription factor (SR11302), and NF-κB and AP-1 (Tanshinone IIA) decreased MMP2 and MMP9 gene expression. NA also enhanced cAMP induction, RAS activation and phosphorylation of ERK1/2. These results suggested that the cAMP-PKA, MAPK, and ROS-NF-κB pathways are involved in β₂-AR signaling. Treatment with 0.1 μM Q3G suppressed ROS generation, cAMP and RAS activation, phosphorylation of ERK1/2 and the expression of HMOX1, MMP2, and MMP9 genes. Furthermore, Q3G (0.1 μM) suppressed invasion of MDA-MB-231 breast cancer cells and MMP-9 induction, and inhibited the binding of [³H]-NA to β₂-AR. These results suggest that Q3G may function to suppress invasion of breast cancer cells by controlling β₂-adrenergic signaling, and may be a dietary chemopreventive factor for stress-related breast cancer.
Quercetin-3-O-glucronide inhibits noradrenaline binding to α<inf>2</inf>-adrenergic receptor, thus suppressing DNA damage induced by treatment with 4-hydroxyestradiol and noradrenaline in MCF-10A cells
2014, Journal of Steroid Biochemistry and Molecular Biology
Citation Excerpt :
Electrochemical detection was performed with a coulometric electrode at 150 mV (for channel 1) and 200 mV (for channel 2). Binding assays for α2-AR were carried out as previously described with some modifications [35–37]. Briefly, cells were seeded at a concentration of 10,000–50,000 cell/well in 10 cm dishes in culture media supplemented with 10% FBS, and incubated at 37 °C for 48–72 h.
Risk factors for breast cancer include estrogens such as 17β-estradiol (E₂) and high stress levels. 4-Hydroxyestradiol (4-OHE₂), a metabolite of E₂ formed preferentially by cytochrome P450 1B1, is oxidized to E₂-3,4-quinone, which reacts with DNA to form depurinating adducts that exert genotoxicity and carcinogenicity. Endogenous catecholamines such as adrenaline (A) and noradrenaline (NA) are released from the adrenal gland and sympathetic nervous system during exposure to stress. Here, we found that treatment with 4-OHE₂ (3 μM) and NA (3 nM) significantly induced the phosphorylation of histone H2AX (γ-H2AX), one of the earliest indicators of DNA damage, and apurinic (AP) sites via the α₂-adrenergic receptor (α₂-AR) in human mammary epithelial MCF-10A cells. As an inverse association between a higher intake of flavonoids and breast cancer risk has previously been suggested from epidemiological studies, we investigated the effects of quercetin-3-O-glucuronide (Q3G), a circulating metabolite of quercetin in the blood, on 4-OHE₂- and NA-induced γ-H2AX and AP sites. Q3G (0.1 μM) suppressed their induction and inhibited the binding of [³H]-NA to α₂-AR. These results suggest that Q3G acts as an α₂-AR antagonist and that it could be used as a chemopreventive agent for stress-promoted breast cancer.
Contribution of α<inf>2</inf>-adrenoceptors to the mitogenic effect of catecholestrogen in human breast cancer MCF-7 cells
2008, Journal of Steroid Biochemistry and Molecular Biology
Catecholestrogens are estrogen metabolites formed by hydroxylation of 17β-estradiol and estrone at either the C-2 or C-4 position, rivaling the parent estrogens in concentration. The objective of the present work was to assess if their catechol group could make them induce proliferation of human breast cancer cells via α₂-adrenoceptors. In competition studies in human breast cancer MCF-7 cells, high concentrations of 2-hydroxy-estradiol (2-OH-E₂), 2-hydroxy-estrone (2-OH-E₁) and 4-hydroxy-estrone (4-OH-E₁) competed for [³H]-rauwolscine binding, whereas 4-hydroxy-estradiol (4-OH-E₂) did not. The contribution of α₂-adrenoceptors and estrogen receptors (ERs) in proliferation enhancement was analyzed with specific antagonists. The specific α₂-adrenergic antagonist yohimbine partially reversed the effect of catecholestrogens except 4-OH-E₂. The selective ER downregulator ICI-182780 or fulvestrant partially or totally reversed the effect of all hydroxylated catecholestrogens. When analyzing the effect of the combination of both antagonists in MCF-7, the contribution of the α₂-adrenoceptors and ERs for 2-OH-E₂, 2-OH-E₁ and 4-OH-E₁ was mixed, whereas for 4-OH-E₂, the only receptor implied was an ER. In MDA-MB-231 cells (ER-α negative) the proliferation stimulation by these three catecholestrogens and reversal by the adrenergic antagonist was also observed. It can be concluded that α₂-adrenoceptors contribute at least in part to the mitogenic effect of 2-OH-E₂, 2-OH-E₁ and 4-OH-E₁.
Potentiation of spinal α<inf>2</inf>-adrenoceptor analgesia in rats deficient in TRPV1-expressing afferent neurons
2007, Neuropharmacology
Citation Excerpt :
Activation of PKC attenuates the inhibitory effect of clonidine on voltage-gated calcium channels (Attali et al., 1991). It has been shown that stimulation of PKC can rapidly desensitize α2-ARs to attenuate the effect of α2-AR agonists (Convents et al., 1989; Jansson et al., 1994). Because of the possible differential expression of PKC in TRPV1- and non-TRPV1-expressing afferent neurons/terminals, this may substantially influence the efficacy of α2-AR agonists.
The α₂-adrenoceptors (α₂-ARs) are located on primary afferent terminals and on neurons in the spinal cord dorsal horn. However, their relative contribution to the analgesic effect of the α₂-AR agonists is not known. In this study, we determined the role of certain presynaptic α₂-ARs in the antinociceptive effect produced by intrathecal administration of the α₂-AR agonist clonidine. TRPV1-expressing sensory neurons were removed by resiniferatoxin (RTX). The effect of intrathecal injection of clonidine was measured by testing the paw withdrawal response to noxious mechanical or heat stimuli. In RTX-treated rats, the α_2A-AR-immunoreactivity co-expressed with TRPV1-expressing terminals in the spinal cord was eliminated. However, the α_2C-AR-immunoreactivity in the spinal cord was little changed. Surprisingly, intrathecal administration of clonidine produced a much greater increase in the mechanical withdrawal threshold in RTX- than in vehicle-treated rats. The duration of the clonidine effect was also significantly increased in RTX-treated rats. Furthermore, in the vehicle-treated group, although intrathecal injection of clonidine produced a large increase in the thermal withdrawal latency, it only had a small and short-lasting effect on the mechanical withdrawal threshold. This study provides new information that the antinociceptive effect of spinally administered α₂-AR agonists is largely modality-specific. Loss of TRPV1-expressing sensory neurons leads to a reduction in presynaptic α_2A-ARs but paradoxically potentiates the effect of clonidine on mechano-nociception.
Different apparent modes of inhibition of α(2A)-adrenoceptor by α<inf>2</inf>-adrenoceptor antagonists
1997, European Journal of Pharmacology
The inhibition of α_2A-adrenoceptor-mediated Ca²⁺ elevation by α₂-adrenoceptor antagonists was measured in HEL human erythroleukemia cells. The antagonists could be divided in two classes: those that displayed surmountable inhibition (right-shift of the agonist dose–response curve), and those that displayed different degrees of insurmountable inhibition (depression of the maximum signal and a possible right-shift of the agonist dose–response curve). The degree of surmountability of the inhibition correlated well with the measured antagonist dissociation rates, suggesting that the hypothesis of the antagonist dissociation rate governing the mode of inhibition of fast responses, holds true. HEL cells thus provide a useful model system for the investigation of physiological consequences of different dissociation rates. Also, the dissociation rates of antagonists not available in radiolabelled form can be predicted from the functional data. The data stresses the importance of measurement of kinetic parameters of the drug–receptor interaction in addition to the equilibrium binding constants.
α<inf>2</inf>-Adrenoceptor regulation of adenylyl cyclase in CHO cells: Dependence on receptor density, receptor subtype and current activity of adenylyl cyclase
1997, European Journal of Pharmacology
Chinese hamster ovary (CHO) cells stably transfected to express different densities of the human α_2A-, α_2B- and α_2C-adrenoceptor subtypes, were used to characterize the regulation of adenylyl cyclase activity by α₂-adrenoceptor agonists. In isolated cell membranes, activation of α_2A- and α_2C-adrenoceptors did not affect basal enzyme activity, but activation of α_2B-adrenoceptors stimulated adenylyl cyclase activity. The extent of stimulation was dependent on the receptor density and was insensitive to pertussis toxin treatment. In the presence of 5 μM forskolin all three receptor subtypes mediated inhibition of adenylyl cyclase activity in a pertussis toxin-sensitive manner. In experiments performed with intact cells the same pattern could be seen: the basal production of cAMP was not affected when α_2C-adrenoceptors were activated, but activated α_2B-adrenoceptors mediated stimulation of cAMP production. In the presence of forskolin, both receptor subtypes mediated inhibition of cAMP production. Our results suggest that α_2B-adrenoceptors are coupled to both G_i and G_s proteins. The signal transduction pathway to which the receptor is coupled is not dependent on receptor density, but its effect on adenylyl cyclase regulation is dependent on the current activity of adenylyl cyclase. The results also suggest that the α_2A- and α_2C-subtypes are preferentially coupled to G_i and transduce only inhibition of adenylyl cyclase activity in transfected CHO cells. At low densities of α_2C-adrenoceptors, clonidine was a partial agonist, but in clones expressing high levels of α_2C-adrenoceptors, clonidine acted as a full agonist by inhibiting cAMP accumulation with the same efficacy as (−)-noradrenaline. This demonstrates that receptor reserve can mask partial agonist activity.

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Regular ArticleDifferent Sensitivity of α2A-C10 and α2C-C4 Receptor Subtypes in Coupling to Inhibition of cAMP Accumulation

Abstract

Regular Article
Different Sensitivity of α₂A-C10 and α₂C-C4 Receptor Subtypes in Coupling to Inhibition of cAMP Accumulation