Alpha2A-adrenoceptors are important modulators of the effects of D-amphetamine on startle reactivity and brain monoamines.

Amphetamines are commonly used to treat attention-deficit hyperactivity disorder, but are also widely abused. They are employed in schizophrenia-related animal models as they disrupt the prepulse inhibition (PPI) of the acoustic startle response. The behavioral effects of amphetamines have mainly been attributed to changes in dopamine transmission, but they also involve increases in the synaptic concentrations of norepinephrine (NE). alpha2-Adrenoceptors (alpha2-ARs) regulate the excitability and transmitter release of brain monoaminergic neurons mainly as inhibitory presynaptic auto- and heteroreceptors. Modulation of acoustic startle and its PPI by the alpha2A-AR subtype was investigated with mice lacking the alpha2A-AR (alpha2A-KO) and their wild-type (WT) controls, without drugs and after administration of the alpha2-AR agonist dexmedetomidine or the antagonist atipamezole. The interaction of D-amphetamine (D-amph) and the alpha2-AR-noradrenergic neuronal system in modulating startle reactivity and in regulating brain monoamine metabolism was assessed as the behavioral and neurochemical responses to D-amph alone, or to the combination of D-amph and dexmedetomidine or atipamezole. alpha2A-KO mice were supersensitive to both neurochemical and behavioral effects of D-amph. Brain NE stores of alpha2A-KO mice were depleted by D-amph, revealing the alpha2A-AR as essential in modulating the actions of D-amph. Also, increased startle responses and more pronounced disruption of PPI were noted in D-amph-treated alpha2A-KO mice. alpha2A-AR also appeared to be responsible for the startle-modulating effects of alpha2-AR drugs, since the startle attenuation after the alpha2-AR agonist dexmedetomidine was absent in alpha2A-KO mice, and the alpha2-AR antagonist atipamezole had opposite effects on the startle reflex in alpha2A-KO and WT mice.     

β-Adrenoceptors potentiate α1-adrenoceptor-mediated inotropic response in rat left atria

AIM: To investigate whether stimulation of β-adrenoceptor (AR) and its subtypes augment α1-AR-evoked positive inotropic response and inositol phosphate (InsP) accumulation in isolated rat left atria. METHODS: Inotropic response was determined by contractile function experiment in isolated electrically driven rat left atria. ^3H-InsP accumulations were measured by ^3H-inositol incorporation and column chromatography. RESULTS: (1) Stimula-tion of α1-AR by phenylephrine (PE) or norepinephrine (NE) in the presence of propranolol (Prop) evoked positive inotropic response and ^3H-InsP accumulations, while stimulation of β-AR by isoprenaline (ISO) or NE in the presence of phentolamine (Phen) only evoked positive inotropic response, but not ^3H-InsP accumulations. (2) Simultaneous stimulation of α1- and β-AR by NE or ISO plus PE significantly shifted the concentration-dependent inotropic response curves and ^3H-InsP accumulation curves to the left and upward compared with individual α1-AR stimulation by PE or NE in the presence of Prop. (3) In the presence of ICI118551 (selective β2-AR antagonist) or CGP12177 (selective β1-AR antagonist), stimulation of either β1- or β2-AR did not change α1-AR-evoked inotropic response and ^3H-InsP accumulations. CONCLUSION: Stimulation of β1-AR and β2-AR potentiates α1-AR-mediated positive inotropic response and InsP accumulation in isolated rat left atria.     

Stimulation of alpha2-adrenoceptors suppresses excitatory synaptic transmission in the medial prefrontal cortex of rat.

Stimulation of alpha2-, especially alpha2A-adrenoceptor (AR), in the prefrontal cortex (PFC) produces a beneficial effect on cognitive functions such as working memory. Alpha2-adrenergic agonists like clonidine and guanfacine have been used experimentally and clinically for treatment of psychiatric disorders such as attention-deficit/hyperactivity disorder (ADHD) and schizophrenia. However, the neurophysiological actions of alpha2-ARs in the PFC are poorly understood. In the present study, we recorded field excitatory post-synaptic potential (fEPSP) and evoked excitatory post-synaptic current (eEPSC) in the medial prefrontal cortex (mPFC) of rats, using in vivo field-potential recording and in vitro whole-cell patch-clamp recording techniques, and examined the effects of the alpha2-AR agonist clonidine and the selective alpha2A-AR agonist guanfacine on fEPSP and eEPSC. Systemic or intra-mPFC application of clonidine or guanfacine significantly reduced fEPSP in the mPFC, either in anesthetized or freely moving rats. Consistently, bath-application of guanfacine suppressed eEPSC in layer V/VI pyramidal neurons, and this effect was blocked by the alpha2-AR antagonist yohimbine or the Gi inhibitor NF023. Moreover, treatment with guanfacine had no effect on paired-pulse facilitation (PPF) of fEPSP and eEPSC. The present study provides the first electrophysiological evidence that stimulation of alpha2A-AR inhibits excitatory synaptic transmission in the mPFC through a post-synaptic mechanism.     

Differential contributions of alpha-1 and alpha-2 adrenoceptors to vasoconstriction in mesenteric arteries and veins of normal and hypertensive mice

Mesenteric veins are more sensitive than arteries to the constrictor effects of sympathetic nerve stimulation and alpha-adrenergic receptor agonists. In the present study, we tested the hypothesis that alpha(2)-adrenergic receptors (alpha(2)-ARs) contribute to in vitro agonist-induced constriction in veins but not arteries and that alpha(2)-AR function is down-regulated in mesenteric arteries and veins in deoxycorticosterone acetate-salt (DOCA-salt) hypertension. Norepinephrine (NE) concentration-response curves were similar in SHAM and DOCA-salt arteries and veins indicating that adrenergic reactivity of mesenteric blood vessels is not altered in murine DOCA-salt hypertension in vitro. Veins were 30-fold more sensitive to NE than arteries. The alpha(1)-AR antagonist, prazosin (0.003-0.3 microM), produced concentration-dependent rightward shifts of the NE concentration-response curves in arteries but not veins. The alpha(2)-AR agonists, clonidine and UK-14,304, did not constrict arteries or veins in the absence or presence of indomethacin (10 microM) and nitro-L-arginine (NLA; 100 microM). The alpha(2)-AR antagonists, yohimbine (0.003-0.3 microM) and rauwolscine (0.1 microM) did not affect NE responses in SHAM or DOCA-salt arteries but antagonized NE responses in veins. These data indicate that there are different alpha-AR contractile mechanisms in murine mesenteric arteries and veins. Alpha(1)-ARs, but not alpha(2)-ARs, mediate direct contractile responses in arteries and veins while alpha(2)-ARs contribute indirectly to NE-induced constrictions in veins but not arteries in vitro. There may be direct protein-protein interactions between alpha(1)- and alpha(2)-ARs or between their signaling pathways in veins. This contribution of alpha(2)-ARs may account for the greater sensitivity of veins compared to arteries to the contractile effects of NE.     

Group II and III metabotropic glutamate receptors suppress excitatory synaptic transmission in the dorsolateral bed nucleus of the stria terminalis.

Conditions such as anxiety, drug abuse, and post-traumatic stress disorder are thought to reflect alterations in central nervous system stress and reward circuitry. Recent evidence suggests a key component of this circuitry is the bed nucleus of the stria terminalis (BNST). In particular, regulation of glutamatergic transmission in the BNST plays a critical role in animal performance on anxiety tasks. Metabotropic glutamate receptors (mGluRs) have been implicated in stress and drug addiction and are known to regulate glutamatergic transmission in many brain regions. We have utilized both extracellular field potential and whole-cell patch-clamp recording in an in vitro slice preparation of mouse dorsal anterolateral BNST to determine whether G(i/o)-linked mGluRs modulate excitatory transmission in this region. We find that activation of group II and group III mGluRs in an in vitro slice preparation of the dBNST causes a depression of excitatory transmission. The depression evoked by group II mGluR activation may represent a form of synaptic plasticity as prolonged activation of the receptor produces a long-term depression of glutamatergic transmission. Based on paired-pulse ratio analysis, initiation of depression by group II and group III mGluR subfamilies appears to, at least in part, involve decreased glutamate release. In total, our data suggest a plausible site of action for some of the anxiolytic effects of group II and group III mGluR agonists.     

Yohimbine Depresses Excitatory Transmission in BNST and Impairs Extinction of Cocaine Place Preference Through Orexin-Dependent,Norepinephrine-Independent Processes

The alpha2 adrenergic receptor (α₂-AR) antagonist yohimbine is a widely used tool for the study of anxiogenesis and stress-induced drug-seeking behavior. We previously demonstrated that yohimbine paradoxically depresses excitatory transmission in the bed nucleus of the stria terminalis (BNST), a region critical to the integration of stress and reward pathways, and produces an impairment of extinction of cocaine-conditioned place preference (cocaine-CPP) independent of α₂-AR signaling. Recent studies show yohimbine-induced drug-seeking behavior is attenuated by orexin receptor 1 (OX₁R) antagonists. Moreover, yohimbine-induced cocaine-seeking behavior is BNST-dependent. Here, we investigated yohimbine-orexin interactions. Our results demonstrate yohimbine-induced depression of excitatory transmission in the BNST is unaffected by alpha1-AR and corticotropin-releasing factor receptor-1 (CRFR₁) antagonists, but is (1) blocked by OxR antagonists and (2) absent in brain slices from orexin knockout mice. Although the actions of yohimbine were not mimicked by the norepinephrine transporter blocker reboxetine, they were by exogenously applied orexin A. We find that, as with yohimbine, orexin A depression of excitatory transmission in BNST is OX₁R–dependent. Finally, we find these ex vivo effects are paralleled in vivo, as yohimbine-induced impairment of cocaine-CPP extinction is blocked by a systemically administered OX₁R antagonist. These data highlight a new mechanism for orexin on excitatory anxiety circuits and demonstrate that some of the actions of yohimbine may be directly dependent upon orexin signaling and independent of norepinephrine and CRF in the BNST.     

Cell proliferation and Ca(2+)-calmodulin dependent protein kinase activation mediated by alpha 1A- and alpha 1B-adrenergic receptor in HEK293 cells

AIM: To examine the ability of alpha 1-AR subtypes on proliferation and Ca(2+)-calmodulin dependent protein kinase (CCDPK, formerly called MAPK) activation in transfected human embryo kidney 293 (HEK293) cells. METHODS: pREP8/alpha 1A-AR, pREP4/alpha 1B-AR, and pREP9/alpha 1D-AR were transfected, respectively, into HEK293 cells by calcium phosphate precipitation. The expression of alpha 1-AR was detected by radioligand binding assays. DNA synthesis was measured by [3H]thymidine incorporation. CCDPK activity was determined by immunoprecipitation method and myelin basic protein was used as substrate. RESULTS: Three clonal HEK293 cell lines stably expressing alpha 1A- or alpha 1B- or alpha 1D-AR were chosen and characterized by radioligand binding assay with receptor densities of about 0.6 nmol.g-1. Treatment with norepinephrine (NE) in the presence of propranolol for 24 h increased DNA synthesis in HEK293/alpha 1A- or HEK293/alpha 1B-AR cells concentration-dependently, with EC50 values of 48.8 nmol.L-1 (95% confidence limits 9.7-246 nmol.L-1) and 8.4 nmol.L-1 (95% confidence limits 2.1-32.9 nmol.L-1), respectively. The increase of DNA synthesis induced by NE 10 mumol.L-1 was 201% +/- 28% and 269% +/- 44% of basal, and the activation of CCDPK was 171% +/- 84% and 292% +/- 92% of basal in HEK293/alpha 1A-AR and HEK293/alpha 1B-AR cells, respectively. Preincubation with prazosin completely abolished NE-induced CCDPK activation in HEK293/alpha 1A- and alpha 1B-AR cells. Those changes were not found in HEK293/alpha 1D-AR cells. CONCLUSION: The activation of alpha 1A- or alpha 1B-AR but not alpha 1D-AR induces cell proliferation.     

Spontaneous activation of beta(2)- but not beta(1)-adrenoceptors expressed in cardiac myocytes from beta(1)beta(2) double knockout mice

Although ligand-free, constitutive beta(2)-adrenergic receptor (AR) signaling has been demonstrated in naive cell lines and in transgenic mice overexpressing cardiac beta(2)-AR, it is unclear whether the dominant cardiac beta-AR subtype, beta(1)-AR, shares the ability of spontaneous activation. In the present study, we expressed human beta(1)- or beta(2)-AR via recombinant adenoviral infection in ventricular myocytes isolated from beta(1)beta(2)-AR double knockout mice, creating pure beta(1)-AR and beta(2)-AR systems with variable receptor densities. A contractile response to a nonselective beta-AR agonist, isoproterenol, was absent in double knockout mouse myocytes but was fully restored after adenoviral beta(1)-AR or adenoviral beta(2)-AR infection. Increasing the titer of adenoviral vectors (multiplicity of infection 10-1000) led to a dose-dependent expression of beta(1)- or beta(2)-AR with a maximal density of 1207 +/- 173 (36-fold over the wild-type control value) and 821+/-38 fmol/mg protein (69-fold), respectively. Using confocal immunohistochemistry, we directly visualized the cellular distribution of beta(1)-AR and beta(2)-AR and found that both subtypes were distributed on the cell surface membrane and transverse tubules, resulting in a striated pattern. In the absence of ligand, beta(2)-AR expression resulted in graded increases in baseline cAMP and contractility up to 428% and 233% of control, respectively, at the maximal beta(2)-AR density. These effects were specifically reversed by a beta(2)-AR inverse agonist, ICI 118,551 (10(-7) M). In contrast, overexpression of beta(1)-AR, even at a greater density, failed to enhance either basal cAMP or contractility; the alleged beta(1)-AR inverse agonist, CGP 20712A (10(-6) M), had no significant effect on basal contraction in these cells. Thus, we conclude that acute beta(2)-AR overexpression in cardiac myocytes elicits significant physiological responses due to spontaneous receptor activation; however, this property is beta-AR subtype specific because beta(1)-AR does not exhibit agonist-independent spontaneous activation.     

Multiple Gi protein subtypes regulate a single effector mechanism.

alpha 2-Adrenergic receptor (alpha 2-AR) responses are mediated by the pertussis toxin-sensitive guanine nucleotide-binding protein (G protein) Gi. Because all three known Gi subtypes are inactivated by pertussis toxin, it has been difficult to determine which of the subtypes are involved in alpha 2-AR responses. In order to investigate alpha 2-AR/Gi coupling, we performed binding and adenylyl cyclase experiments in membranes from CHO-K1 cells transfected with the human alpha 2A-AR. Antisera directed against the carboxyl-terminal region of the Gi1/Gi2 or the Gi3 proteins were used to determine which subtypes were important for high affinity agonist binding and inhibition of adenylyl cyclase. The CHO-K1 cell membranes exhibited immunoreactivity at an apparent molecular mass of 40-41 kDa for both Gi1/Gi2 and Gi3 antisera. Western blot analysis, using purified bovine brain G proteins for comparison, demonstrated that the transfected CHO-K1 cells possess Gi2 and Gi3. High affinity guanosine 5'-(beta,gamma-imido) triphosphate-sensitive binding of the alpha 2-AR agonists [3H]bromoxidine and p-[125I]iodoclonidine ([125I]PIC) was reduced by 30-50% by either the Gi1/Gi2 or Gi3 antiserum. Bromoxidine (1 microM) and PIC (1 microM) inhibited membrane adenylyl cyclase by 34 and 27%, respectively. Gi3 antiserum reduced the inhibition by 26% and 67% for bromoxidine and PIC, respectively. The Gi1/Gi2 antiserum reduced the inhibition by 56% and 63% for bromoxidine and PIC, respectively. Furthermore, when both antisera were used together, there was a complete reversal of alpha 2-AR-mediated inhibition. These observations provide evidence of alpha 2A-AR coupling to at least two subtypes of Gi proteins and the first evidence of functional involvement of Gi3 in the inhibition of adenylyl cyclase.     

Norepinephrine induces lipolysis in beta1/beta2/beta3-adrenoceptor knockout mice

Catecholamines are major stimulants of adipose tissue metabolism. Norepinephrine and epinephrine act through three subtypes of beta-adrenoceptors (beta-AR) expressed in the adipocytes. The aim of this work was to study the mechanisms of lipid mobilization in beta1/beta2/beta3-AR triple-knockout (beta-less) mice. Glycerol and nonesterified fatty acids released from isolated adipocytes were measured as an index of lipolytic activity. There was no difference between the two genotypes for basal lipolysis and lipolytic response to corticotropin or to agents acting at the adenylyl cyclase and protein kinase A levels. The lipolytic response to norepinephrine and beta-AR agonists was blunted in beta-less mice. However, a residual low-affinity lipolytic effect was observed in the presence of catecholamines and beta3-AR agonists but not of beta1- or beta2-AR agonists. cAMP levels were increased by a beta-AR agonist in white and brown adipocytes of beta-less mice. The residual lipolytic effect was blocked by beta-AR antagonists. It was mediated neither by alpha1- or alpha2-AR nor dopaminergic, serotonergic, and histaminergic by receptors. Bioinformatic analyses do not provide evidence for a fourth beta-AR. We conclude that the residual lipolytic effect observed in beta-less mice can be attributed to an unknown Gs-protein-coupled receptor with low affinity for catecholamines.     

Alpha 2-adrenergic agonist enrichment of spinophilin at the cell surface involves beta gamma subunits of Gi proteins and is preferentially induced by the alpha 2A-subtype

Agonist activation regulates reciprocal interactions of spinophilin and arrestin with the alpha2A- and alpha2B -adrenergic receptor (AR) subtypes via their 3i loop. Because arrestin association with G protein-coupled receptor is preceded by redistribution of arrestin to the cell surface, the present studies explored whether agonist activation of the alpha2A- and alpha2B -AR subtypes also led to spinophilin enrichment at the cell surface. Live cell imaging studies using a green fluorescent protein-tagged spinophilin examined spinophilin localization and its regulation by alpha2 -AR agonist. Agonist activation of alpha2A-AR preferentially, compared with the alpha2B-AR, led to spinophilin enrichment at the cell surface in human embryonic kidney 293 cells and in mouse embryo fibroblasts derived from spinophilin null mice. Activation of the delta LEESSSS alpha2A-AR, which has enriched association with spinophilin compared with the wild-type (WT) alpha2A-AR, does not show an enhanced redistribution of spinophilin to the surface compared with WT alpha2A-AR, demonstrating that the ability or affinity of the receptor in binding spinophilin may be independent of the ability of the receptor to effect spinophilin redistribution to the surface. Agonist-evoked enrichment of spinophilin at the cell surface seems to involve downstream signaling events, manifested both by the pertussis toxin sensitivity of the process and by the marked attenuation of spinophilin redistribution in cells expressing the beta-adrenergic receptor kinase-C tail, which sequesters beta gamma subunits of G proteins. Together, the data suggest that agonist-evoked spinophilin enrichment at the cell surface is caused by receptor-evoked signaling pathways and is independent of the affinity of the receptor for the spinophilin molecule.     

Differential regulation of the cell-surface targeting and function of beta- and alpha1-adrenergic receptors by Rab1 GTPase in cardiac myocytes

The molecular mechanism underlying the export from the endoplasmic reticulum (ER) to the cell surface and its role in the regulation of signaling of adrenergic receptors (ARs) remain largely unknown. In this report, we determined the role of Rab1, a Ras-like GTPase that coordinates protein transport specifically from the ER to the Golgi, in the cell surface targeting and function of endogenous beta- and alpha1-ARs in neonatal rat ventricular myocytes. Adenovirus-driven expression of Rab1 into myocytes selectively increased the cell-surface number of alpha1-AR, but not beta-AR, whereas the dominant-negative mutant Rab1N124I significantly reduced the cell-surface expression of beta-AR and alpha1-AR. Brefeldin A inhibited beta-AR and alpha1-AR export and antagonized the Rab1 effect on alpha1-AR expression. Manipulation of Rab1 function similarly influenced the transport of alpha1A- and alpha1B-ARs as well as beta1- and beta2-ARs. Fluorescent microscopy analysis demonstrated that expression of Rab1N124I and Rab1 small interfering RNA induced a marked accumulation of GFP-tagged beta2-AR and alpha1B-AR in the ER. Consistent with the effects on receptor cell-surface targeting, Rab1 selectively enhanced ERK1/2 activation and hypertrophic growth in response to the alpha1-AR agonist phenylephrine but not to the beta-AR agonist isoproterenol. Rab1N124I inhibited both agonist-mediated ERK1/2 activation and hypertrophic growth in neonatal myocytes. These results demonstrate that the cell-surface targeting and signaling of beta- and alpha1-ARs require Rab1 and are differentially modulated by augmentation of Rab1 function. Our data provide strong evidence implicating the ER-to-Golgi traffic as a site for selective manipulation of distinct AR function in cardiac myocytes.     

Functional α1-adrenergic receptor subtypes in human right gastroepiploic artery

AIM: To study the functional alpha1-adrenergic receptor (alpha1-AR) subtypes in human right gastroepiploic artery (RGA). METHODS: The effects of alpha2-AR, alpha1-AR, and alpha1-AR subtype selective antagonists on norepinephrine (NE)-induced vasoconstriction in isolated human RGA were observed by contractile function experiment. RESULTS: Cumulative concentration-response curves for NE were competitively antagonized in RGA by alpha2-AR selective antagonist yohimbine (pA2 6.82+/-0.28, slope 1.12+/-0.40),alpha1-AR selective antagonist prazosin (pA2 9.77+/-0.22, slope 0.90+/-0.22),alpha1A-AR selective antagonists RS17053 (pA2 8.42+/-0.20, slope 0.93+/-0.20) and 5-MU (pA2 8.42+/-0.22, slope 0.88+/-0.18),alpha1D-AR selective antagonist BMY7378 (pA2 6.84+/-0.32, slope 1.05+/-0.17), and alpha1A-,alpha1B-AR selective antagonist WB4101 (pA2 8.88+/-0.20, slope 1.15+/-0.16). The correlation coefficients between these pA2 values of alpha1-AR selective antagonists with pKi values of which obtained from alpha1A-, alpha1B- and alpha1D-AR cloned cells are 0.95, 0.82, and 0.42. After the vessels were pretreated by chlorethylclonidine (CEC), an alpha1B- and alpha1D-AR irreversible alkylating agent, the pD2 values were changed from 5.9+/-0.5 to 5.6+/-0.6 and the maximal contraction was changed from (8.9+/-3.2) g to (8.0+/-3.2) g, respectively. The difference was not significant. CONCLUSION: In human RGA, the contraction response is mainly mediated by alpha1-AR, of which alpha1A-AR plays an important role, whereas alpha1B- and alpha1D-AR are not involved in the contraction response.     

一种新的α1A肾上腺素受体选择性拮抗剂—Sertindole

本工作分别在稳定表达α_1A,α_1B和α_1D肾上腺素受体(adrenoceptor,AR)的人胚胎肾脏细胞( human embryonic kidney 293,HEK 293)和大鼠离体血管上,用放射配体结合实验和离体血管收缩功能实验方法以确定sertindole对α₁-AR亚型的选择性拮抗作用。结果显示sertindole与克隆α_1A-AR的亲和性分别是与克隆α_1B-AR和克隆α_1D-AR的69倍和132倍。Sertindole拮抗去甲肾上腺素引起的主动脉和肾动脉收缩反应的pA₂值分别与其对α_1D和α_1A亚型的pKI值相符。分别稳定表达3种亚型受体的HEK293细胞膜标本经与sertindole预温育30min后,受体与¹²⁵IBE2254结合的B_max值显著降低,KD值无显著变化;而在 sertindole 存在条件下,α₁-AR3种亚型与¹²⁵IBE2254 结合的KD值显著增大,但B_max值无显著改变。上述结果表明sertindole为不可逆性竞争性α₁-AR拮抗剂,并有α_1A亚型选择性。     

The Janus faces of adrenoceptors: factors controlling the coupling of adrenoceptors to multiple signal transduction pathways

1. The adrenoceptors (AR) are an important subfamily of rhodopsin-like G-protein-coupled receptors that couple to an increasingly large number of signalling mechanisms. Two important factors that determine the pathways that are used are the C-terminal region of the receptor and the agonist used to activate the receptor. 2. Studies of splice variants of the mouse beta3-AR showed that the C-terminus is a factor controlling the signalling characteristics. Although these receptors differ only at the C-terminus, the beta3b-AR coupled to both Gs and Gi, whereas the beta3a-AR coupled solely to Gs. 3. Examination of four splice variants of the human alpha1A-AR showed that all were able to couple to pertussis toxin-sensitive G-proteins, even though they have radically different C-terminal regions. 4. Comparison of the effects of the beta3-AR ligands CL316243 and SR59230A showed that both can activate the mouse beta3-AR but that SR59230A uses pathways other than cAMP accumulation in 3T3-F442A cells. 5. Examination of a series of alpha1-AR agonists for their ability to activate a number of signalling pathways revealed that A61603 acted as a full agonist in all assays, whereas oxymetazoline was unable to cause cAMP accumulation, suggesting agonist-selective signalling at the human alpha1A-AR.     

Desensitization of alpha 2A-adrenoceptor signalling by modest levels of adrenaline is facilitated by beta 2-adrenoceptor-dependent GRK3 up-regulation

(1) Adrenaline (ADR) and noradrenaline (NA) can simultaneously activate inhibitory alpha(2)- and stimulatory beta-adrenoceptors (AR). However, ADR and NA differ significantly in that ADR is a potent beta(2)-AR agonist while NA is not. Only recently has the interaction resulting from the simultaneous activation of alpha(2)- and beta(2)-AR been examined at the cellular level to determine the mechanisms of alpha(2)-AR regulation following concomitant activation of both alpha(2)- and beta(2)-ARs by chronic ADR. (2) This study evaluates beta(2)-AR regulation of alpha(2A)-AR signalling following chronic ADR (300 nM) and NA (1 and 30 micro M) treatments of BE(2)-C human neuroblastoma cells that natively express both beta(2)- and alpha(2A)-ARs. (3) Chronic (24 h) treatment with ADR (300 nM) desensitized the response to the alpha(2A)-AR agonist, brimonidine, in BE(2)-C cells. Addition of the beta-AR antagonist, propranolol, blocked the ADR-induced alpha(2A)-AR desensitization. Unlike ADR, chronic NA (1 micro M) treatment had no effect on the alpha(2A)-AR response. However if NA was increased to 30 micro M for 24 h, alpha(2A)-AR desensitization was observed; this desensitization was partially reversed by propranolol. (4) Chronic ADR (300 nM) treatment reduced alpha(2A)-AR binding levels, contributing to the alpha(2A)-AR desensitization. This decrease was prevented by addition of propranolol during ADR treatment. Chronic NA (30 micro M), like ADR, treatment lowered specific binding, whereas 1 micro M NA treatment was without effect. (5) Chronic ADR treatment produced a significant increase in GRK3 levels and this was blocked by propranolol or GRK2/3 antisense DNA treatment. This antisense DNA, common to both GRK2 and GRK3, also blocked chronic ADR-induced alpha(2A)-AR desensitization and down-regulation. (6) Acute (1 h) ADR (300 nM) or NA treatment (1 micro M) produced alpha(2A)-AR desensitization. The desensitization produced by acute treatment was beta-AR independent, as it was not blocked by propranolol. (7) We conclude that chronic treatment with modest levels of ADR produces alpha(2A)-AR desensitization by mechanisms that involve up-regulation of GRK3 and down-regulation of alpha(2A)-AR levels through interactions with the beta(2)-AR.     

Alpha-receptor-mediated facilitation of somatosensory cortical neuronal responses to excitatory synaptic inputs and iontophoretically applied acetylcholine

The major objective of the present study was to characterize, in terms of α- and β-receptor mechanisms, the previously observed facilitating actions of norepinephrine (NE) on somatosensory cortical neuronal responses to excitatory synaptic inputs and iontophoretic administration of acetylcholine (ACh). In the forelimb region of rat somatosensory cortex (SI), excitatory single unit responses to natural stimulation of the contralateral forepaw or microiontophoretic pulses (10 sec duration at 45 sec intervals) of ACh were examined before, during and after iontophoretic administration of NE, phenylephrine (PE) and isoproterenol (ISO). Adrenergic agonist actions were quantitatively assessed by computer-based analysis of poststimulus time or cholinergic response histograms. At doses which suppressed or had no effect on the spontaneous discharge, the α agonist, phenylephrine, facilitated excitatory synaptic transmission in 12 of 14 cells and enhanced neuronal responsiveness to ACh in 15 of 24 cells. Thus, phenylephrine consistently mimicked the potentiative actions of NE. whereas the β agonist, isoproterenol, at similar doses, was ineffective in enhancing synaptic efficacy (n = 12) or ACh-induced excitation (n = 19). In addition, iontophoretic application of the α blocker, phentolamine. reversibly antagonized NE-induced potentiation of ACh responses in 6 out of 6 cases. In contrast, administration of the beta antagonist, sotalol, had no effect on NE-mediated enhancement of ACh in all three neurons tested. Overall, these results suggest that endogenously released NE may facilitate excitatory synaptic transmission within the somatosensory cortex by activation of postsynaptic adrenoceptors with α characteristics.     

Role of G(i)-proteins in norepinephrine-mediated vasoconstriction in rat tail artery smooth muscle

We showed, in rat de-endothelialised tail artery, that pertussis toxin (PTX) (1 microg/mL, 2 hr) attenuated norepinephrine (NE)-induced vasoconstriction without modifying intracellular calcium concentration [Ca2+](i) mobilisation. We suggested the existence of two NE-induced intracellular pathways: a first, which would be insensitive to PTX and lead to [Ca2+](i) mobilisation, and a second sensitive to PTX and involved in the [Ca2+](i) sensitivity of NE-induced contraction. The aim of this study was to demonstrate the existence of the second intracellular pathway. PTX-sensitive G(i/o)-proteins in rat tail artery SMC membrane were identified by immunoblot and ADP-ribosylation. [(32)P]ADP-ribosylation of alpha(i/o)-subunits was demonstrated in situ by perfusing rat de-endothelialised tail artery segments with PTX (1 microg/mL, 2 hr), which suggested that G(i/o)-protein inactivation was involved in the reduction by PTX of the [Ca2+](i) sensitivity of NE-induced contraction. Coupling between G(i/o)-proteins and NE receptors was confirmed by the NE-induced increase in G(i/o)-specific GTPase activity (24.1 +/- 1.9 vs 8.8 +/- 0.4 pmol P(i)/mg protein at 5 min; P < 0.05 vs basal). [(3)H]Prazosin-binding data showed the presence of a heterogeneous alpha(1)-AR population in rat tail artery smooth muscle cells. We demonstrated the in vitro coupling between alpha(1A)-AR subtype and alpha(i)-subunits. In conclusion, we identified, in rat de-endothelialised tail artery, a PTX-sensitive G(i/o)-protein-modulated pathway that is coupled to NE receptors via alpha(1A)-AR. We suggest that NE stimulates two alpha(1)-AR-mediated intracellular pathways: a first, which is mediated by a G(q)-protein and leads to [Ca2+](i) mobilisation and contraction, and a second, which is mediated by a G(i)-protein and is involved in the amplification of the [Ca2+](i) sensitivity of NE-induced tension.     

Characterization of cAMP accumulation mediated by three α1—adrenoceptor subtypes in HEK293 cells

AIM:To investigate the characterization of cAMP response mediated by α1-adrenoceptor (α1-AR) subtypes in HEK293 cells. METHODS:(1) Full-length cDNA encoding three α1-AR subtypes were transfected into HEK293 cells by the calcium phosphate precipitation method, respectively. (2) The densities of α1-AR subtypes expressed in HEK293 cells were measured by radioligand binding assay. (3)cAMP accumulation was measured by [^3H] adenine prelabeling method. RESULTS: (1)Activation of each of three subtypes resulted in an increase of cAMP accumulation in HEK293 cells in a dose-dependent manner, which was inhibited by selective α1-AR antagonist prazosin. (2) Comparing the pharmacological property, the maximal responses of α1A-AR to agonists were the most potent, while the sensitivity of α1-AR subtypes to norepinephrine(NE) was the highest. CONCLUSION: Each of three α1-AR subtypes can mediate cAMP accumulation in HEK293 cell line, and there are differences in pharmacological property.     

Receptor subtype involved in α1-adrenergic receptor-mediated Ca^2＋ signaling in cardiomyocytes

Aim： The enhancement of intracellular Ca^＋signaling in response to α1-adrenergic receptor （α1-AR） stimulation is an essential signal transduction event in the regulation of cardiac functions, such as cardiac growth, cardiac contraction, and cardiac adaptation to various situations. The present study was intended to determine the role（s） of the α1-AR subtype（s） in mediating this response. Methods： We evaluated the effects of subtype-specific agonists and antagonists of the α1- AR on the intracellular Ca^2＋ signaling of neonatal rat ventricular myocytes using a confocal microscope. Results： After being cultured for 48 h, the myocytes exhibited spontaneous local Ca^2＋ release, sparks, and global Ca^2＋ transients. The activation of the α1-AR with phenylephrine, a selective agonist of the α1-AR, dose-dependently increased the frequency of Ca^2＋ transients with an EC5o value of 2.3 larnol/L. Blocking the α1A-AR subtype with 5-methylurapidil （5-Mu） inhi- bited the stimulatory effect of phenylephrine with an IC50 value of 6.7 nmol/L. In contrast, blockade of the α1B-AR and α1D-AR subtypes with chloroethylclonidine and BMY 7378, respectively, did not affect the phenylephrine effect. Similarly, the local Ca^2＋ spark numbers were also increased by the activation of the α1-AR, and this effect could be abolished selectively by 5-Mu. More importantly, A61603, a novel selective α1A-AR agonist, mimicked the effects of phenylephrine, but with more potency （EC50 value =6.9 nmol/L） in the potentiation of Ca^2＋ transients, and blockade of the α1A-AR by 5-Mu caused abolishment of its effects. Conclusion： These results indicate that α1-adrenergic stimulation of intracellular Ca^2＋ activity is mediated selectively by the α1-AR.