Simultaneous alpha2B- and beta2-adrenoceptor activation sensitizes the alpha2B-adrenoceptor for agonist-induced down-regulation

We recently reported that alpha(2A)-adrenoceptor (AR) desensitization and down-regulation occurs after 24-h treatment with epinephrine (EPI) (0.3 microM) in BE(2)-C cells that express both alpha(2)- and beta(2)-ARs. The same concentration of norepinephrine (NE) has no effect. The effect of EPI is prevented by beta(2)-AR blockade and is associated with an increase in G protein-coupled receptor kinase 3 (GRK3) expression. Because differences in agonist-induced down-regulation of the alpha(2A)-versus alpha(2B)-ARs have been reported, the present study examines the effects of simultaneous activation of alpha(2B)- and beta(2)-ARs on alpha(2B)-AR number and signaling. We studied NG108 cells that naturally express alpha(2B)-ARs, and BN17 cells, NG108 cells transfected to express the human beta(2)-AR. In NG108 cells, alpha(2B)-AR desensitization and down-regulation require treatment with 20 microM EPI or NE; GRK expression was not changed. In BN17 cells expressing beta(2)-ARs, the threshold EPI concentration for alpha(2B)-AR desensitization and down-regulation was reduced to 0.3 microM; 10 microM NE was required for the same effect. Furthermore, 24-h EPI or NE treatments that produced desensitization also resulted in a selective 2-fold up-regulation of GRK3; GRK2 was unchanged. The beta-AR antagonist alprenolol (1 microM) and GRK3 antisense (but not sense) DNA blocked 0.3 microM EPI- and 10 microM NE-induced desensitization and down-regulation of the alpha(2B)-AR as well as GRK3 up-regulation. In conclusion, simultaneous activation of alpha(2B)- and beta(2)-ARs results in a 67-fold decrease in the threshold concentration of EPI required for alpha(2B)-AR down-regulation. This lower threshold for down-regulation is associated with alpha(2B)- and beta(2)-AR dependent up-regulation of GRK3 expression.     

Alpha(2)-adrenergic receptor signalling in hypertension

Cardiovascular regulation by the sympathetic nervous system is mediated by activation of one or more of the nine known subtypes of the adrenergic receptor family; alpha(1A)-, alpha(1B)-, alpha(1D)-, alpha(2A)-, alpha(2B)-, alpha(2C)-, beta(1)-, beta(2)- and beta(3)-ARs (adrenoceptors). The role of the alpha(2)-AR family has long been known to include presynaptic inhibition of neurotransmitter release, diminished sympathetic efferent traffic, vasodilation and vasoconstriction. This complex response is mediated by one of three subtypes which all uniquely affect blood pressure and blood flow. All three subtypes are present in the brain, kidney, heart and vasculature. However, each differentially influences blood pressure and sympathetic transmission. Activation of alpha(2A)-ARs in cardiovascular control centres of the brain lowers blood pressure and decreases plasma noradrenaline (norepinephrine), activation of peripheral alpha(2B)-ARs causes sodium retention and vasoconstriction, whereas activation of peripheral alpha(2C)-ARs causes cold-induced vasoconstriction. In addition, non-selective agonists elicit endothelium-dependent vasodilation and presynaptic inhibition of noradrenaline release. The evidence that each of these receptor subtypes uniquely participates in cardiovascular control is discussed in this review.     

Alpha1-adrenergic receptors activate Ca(2+)-permeable cationic channels in prostate cancer epithelial cells

The prostate gland is a rich source of alpha1-adrenergic receptors (alpha1-ARs). alpha1-AR antagonists are commonly used in the treatment of benign prostatic hyperplasia symptoms, due to their action on smooth muscle cells. However, virtually nothing is known about the role of alpha1-ARs in epithelial cells. Here, by using two human prostate cancer epithelial (hPCE) cell models - primary cells from resection specimens (primary hPCE cells) and an LNCaP (lymph node carcinoma of the prostate) cell line - we identify an alpha1A subtype of adrenergic receptor (alpha1A-AR) and show its functional coupling to plasmalemmal cationic channels via direct diacylglycerol (DAG) gating. In both cell types, agonist-mediated stimulation of alpha1A-ARs and DAG analogues activated similar cationic membrane currents and Ca(2+) influx. These currents were sensitive to the alpha1A-AR antagonists, prazosin and WB4101, and to transient receptor potential (TRP) channel blockers, 2-aminophenyl borate and SK&F 96365. Chronic activation of alpha1A-ARs enhanced LNCaP cell proliferation, which could be antagonized by alpha1A-AR and TRP inhibitors. Collectively, our results suggest that alpha1-ARs play a role in promoting hPCE cell proliferation via TRP channels.     

S18616, a highly potent spiroimidazoline agonist at alpha(2)-adrenoceptors: II. Influence on monoaminergic transmission, motor function, and anxiety in comparison with dexmedetomidine and clonidine

The alpha(2)-adrenoceptor (AR) agonist, S18616 ((S)-spiro[(1-oxa-2-amino-3-azacyclopent-2-ene)-4,2'-(8'-chloro-1' , 2',3',4'-tetrahydronaphthalene)] accompanying article), suppressed electrical activity of adrenergic neurons in the locus ceruleus, an action reversed by the alpha(2)-AR antagonist, idazoxan, which itself enhanced their firing rate. Electrical activity of serotonergic neurons in the dorsal raphe nucleus was similarly suppressed, an action likewise blocked by idazoxan, which did not, itself, influence firing. In freely moving rats, S18616 decreased extracellular levels of norepinephrine (NE), serotonin (5-HT), and dopamine (DA) in frontal cortex and hippocampus. The selective alpha(2)- versus alpha(1)-AR antagonists, atipamezole and BRL-44408 (a preferential alpha(2A)-AR antagonist), elevated levels of NE and DA but not 5-HT. In their presence, the influence of S18616 on frontocortical levels of NE, DA, and 5-HT was blocked. In contrast, prazosin, a selective alpha(1)- versus alpha(2)-AR antagonist (which also preferentially blocks alpha(2B/2C)-ARs) dose dependently decreased levels of 5-HT, but not NE and DA, and failed to modify the actions of S18616. Ultrasonic vocalizations elicited by rats in an aversive environment were inhibited by S18616, which also suppressed aggressive and marble-burying behaviors in mice. Furthermore, S18616 (biphasically) enhanced punished responses in the Vogel conflict test and active social interaction tests in rats. At higher doses, S18616 displayed sedative/hypnotic properties. Both anxiolytic and motor actions of S18616 were inhibited by atipamezole and BRL-44408 but not prazosin. Dexmedetomidine mimicked the actions of S18616 at higher doses except for more potent sedative/hypnotic properties. Clonidine also mimicked S18616, but only at markedly higher doses. In conclusion, via activation of alpha(2)-ARs, S18616 potently inhibits corticolimbic adrenergic, serotonergic, and (frontocortical) dopaminergic transmission in parallel with the expression of its anxiolytic and sedative properties.     

Tonic inhibitory role for cAMP in alpha(1a)-adrenergic receptor coupling to extracellular signal-regulated kinases 1/2

alpha(1a)-Adrenergic receptors (ARs) couple to phosphoinositide hydrolysis, adenylyl cyclase, and mitogen-activated protein kinase (MAPK) pathways. However, the interaction among these signaling pathways in activating extracellular signal-regulated kinase 1/2 (ERK1/2) is not well understood. We investigated the coupling of alpha(1a)-ARs to ERK1/2 in Chinese hamster ovary (CHO)-K1 cells stably transfected with mouse alpha(1a)-ARs, as well as the interaction between ERK1/2 and norepinephrine-induced cAMP accumulation. alpha(1a)-AR activation by norepinephrine increased the cytosolic Ca(2+) concentration and phosphorylated ERK1/2 in a time- and concentration-dependent manner. ERK1/2 phosphorylation was blocked by the MAPK kinase 1/2 inhibitor 2'-amino-3'-methoxyflavone (PD 98059) and the alpha(1)-AR antagonist prazosin. A transient elevation in intracellular Ca(2+) was required for the phosphorylation of ERK1/2; however, activation of protein kinase C did not seem to be required for ERK1/2 phosphorylation. Norepinephrine also stimulated cAMP accumulation in transfected CHO-K1 cells in a concentration-dependent manner via alpha(1a)-ARs, which was blocked by the Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Norepinephrine-induced ERK1/2 phosphorylation was inhibited by the adenylyl cyclase activator forskolin and was enhanced by the adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purine-6-amine (SQ 22536) and the protein kinase A inhibitor 4-cyano-3-methylisoquinoline. In conclusion, in transfected CHO-K1 cells, alpha(1a)-AR activation activates both phospholipase C and adenylyl cyclase-mediated signaling pathways. alpha(1a)-AR-mediated ERK1/2 phosphorylation was dependent on a rise in intracellular Ca(2+), and this pathway was reciprocally regulated by the concomitant activation of adenylyl cyclase, which inhibits ERK1/2 phosphorylation. Thus, alpha(1a)-AR stimulation of cAMP production may play an important role in regulating ERK1/2 phosphorylation in cell lines and native tissues.     

Binding, partial agonism, and potentiation of alpha(1)-adrenergic receptor function by benzodiazepines: A potential site of allosteric modulation

Benzodiazepines, a class of drugs commonly used to induce anesthesia and sedation, can attenuate intracellular calcium oscillations evoked by alpha(1)-adrenergic receptor (alpha(1)-AR) stimulation in pulmonary artery smooth muscle cells. We postulated a direct action of benzodiazepines in modulating alpha(1)-AR function at the receptor level. Benzodiazepines bound to each of the cloned alpha(1)-AR subtypes (alpha(1a)-, alpha(1b)-, or alpha(1d)-AR) on COS-1 cell membranes transiently transfected to express a single population of alpha(1)-AR subtype. The ability of benzodiazepines to alter alpha(1)-AR signal transduction was investigated by measuring total inositol phosphate generation in rat-1 fibroblast cells, stably transfected to express a single alpha(1)-AR subtype. By themselves, benzodiazepines displayed partial agonism. At alpha(1b)-ARs and alpha(1d)-ARs, the maximal inositol phosphate response to phenylephrine was potentiated almost 2-fold by either midazolam or lorazepam (100 microM). At alpha(1a)-ARs, diazepam, lorazepam, and midazolam all increased the maximal response of the partial agonist clonidine at these receptors, whereas the response to the full agonist phenylephrine was unaltered or inhibited. The potentiating actions of midazolam and its partial agonism at alpha(1)-ARs was blocked by the addition of 1 microM prazosin, an alpha(1)-AR antagonist, and not by a gamma-aminobutyric acid(A)-receptor antagonist. These studies show that benzodiazepines modulate the function of alpha(1)-ARs in vitro, and this is the first report of a potential allosteric site on alpha(1)-ARs that may be therapeutically useful for drug design.     

S18327 (1-[2-[4-(6-fluoro-1, 2-benzisoxazol-3-yl)piperid-1-yl]ethyl]3-phenyl imidazolin-2-one), a novel, potential antipsychotic displaying marked antagonist properties at alpha(1)- and alpha(2)-adrenergic receptors: I. Receptorial, neurochemical, and electrophysiological profile

S18327 displayed modest affinity for human (h)D(2) and hD(3) receptors and high affinity for hD(4) receptors. At each, S18327 antagonized stimulation of [(35)S]guanosine-5'-O-(3-thio)triphosphate binding by dopamine (DA). It also blocked activation of mitogen-activated protein kinase at hD(3) receptors. The affinity of S18327 at hD(1) and hD(5) sites was modest. S18327 showed pronounced affinity for human serotonin (h5-HT)(2A) receptors and human alpha(1A)-adrenergic receptors (hARs), at which it antagonized increases in intracellular Ca(2+) concentration levels elicited by 5-HT and norepinephrine (NE), respectively. S18327 presented significant affinity for halpha(2A)-ARs and antagonized NE-induced[(35)S]guanosine-5'-O-(3-thio)triphosphate binding both at these sites and at alpha(2)-ARs in rat amygdala. Reflecting blockade of alpha(2)-autoreceptors, S18327 enhanced firing of adrenergic neurons in locus ceruleus, accelerated hippocampal synthesis of NE, and increased dialysate levels of NE in hippocampus, accumbens, and frontal cortex. S18327 abolished inhibition of ventrotegmental area-localized dopaminergic neurons by apomorphine. However, S18327 alone did not affect their activity and only modestly enhanced cerebral turnover of DA and dialysate levels of DA in striatum and accumbens. In contrast, S18327 markedly increased dialysate levels of DA in frontal cortex, an action abolished by the selective alpha(2)-AR agonist, S18616. Finally, S18327 reduced synthesis and dialysate levels of 5-HT in striatum and suppressed firing of dorsal raphe-localized serotonergic neurons, an action attenuated by the alpha(1)-AR agonist cirazoline. In conclusion, S18327 possesses marked antagonist activity at alpha(1)-ARs and D(4) and 5-HT(2A) receptors and less potent antagonist activity at alpha(2)-ARs and D(1) and D(2) receptors. Antagonism by S18327 of alpha(2)-ARs enhances adrenergic transmission and reinforces frontocortical dopaminergic transmission, whereas blockade of alpha(1)-ARs inhibits dorsal raphe-derived serotonergic pathways. As further described in the accompanying paper, this profile of activity may contribute to the potential antipsychotic properties of S18327.     

Antiparkinsonian agent piribedil displays antagonist properties at native, rat, and cloned, human alpha(2)-adrenoceptors: cellular and functional characterization

Compared with cloned, human (h)D(2) receptors (pK(i) = 6.9), the antiparkinsonian agent piribedil showed comparable affinity for halpha(2A)- (7.1) and halpha(2C)- (7.2) adrenoceptors (ARs), whereas its affinity for halpha(2B)-ARs was less marked (6.5). At halpha(2A)- and halpha(2C)-ARs, piribedil antagonized induction of [(35)S]guanosine-5'-O-(3-thio)triphosphate (GTPgammaS) binding by norepinephrine (NE) with pK(b) values of 6.5 and 6.9, respectively. Furthermore, Schild analysis of the actions of piribedil at halpha(2A)-ARs indicated competitive antagonism, yielding a pA(2) of 6.5. At a porcine alpha(2A)-AR-Gi1alpha-Cys351C (wild-type) fusion protein, piribedil competitively abolished (pA(2) = 6.5) GTPase activity induced by epinephrine. However, at a alpha(2A)-AR-Gi1alpha-Cys351I (mutant) fusion protein of amplified sensitivity, although still acting as a competitive antagonist (pA(2) = 6.2) of epinephrine, piribedil itself manifested weak partial agonist properties. Similarly, piribedil weakly induced mitogen-activated protein kinase phosphorylation via wild-type halpha(2A)-ARs, although attenuating its phosphorylation by NE. As demonstrated by functional [(35)S]GTPgammaS autoradiography in rats, piribedil antagonized activation by NE of alpha(2)-ARs in cortex, amygdala, and septum. Antagonist properties were also expressed in a dose-dependent enhancement of the firing rate of adrenergic neurons in locus ceruleus (0.125-4.0 mg/kg i.v.). Furthermore, piribedil (2.5-4.0 mg/kg s.c.) accelerated hippocampal NE synthesis, elevated dialysis levels of NE in hippocampus and frontal cortex, and blocked hypnotic-sedative properties of the alpha(2)-AR agonist xylazine. Finally, piribedil showed only modest affinity for rat alpha(1)-ARs (5.9) and weakly antagonized NE-induced activation of phospholipase C via halpha(1A)-ARs (pK(b) = 5.6). In conclusion, piribedil displays essentially antagonist properties at cloned, human and cerebral, rat alpha(2)-ARs. Blockade of alpha(2)-ARs may, thus, contribute to its clinical antiparkinsonian profile.     

alpha(1B)- and alpha(1D)-Adrenergic receptors exhibit different requirements for agonist and mitogen-activated protein kinase activation to regulate growth responses in rat 1 fibroblasts.

We compared DNA replication, protein biosynthesis, and mitogen-activated protein kinase (MAPK) activity in Rat 1 fibroblasts stably expressing either the alpha(1B)-adrenergic receptor (AR) or alpha(1D)-AR subtypes. Activation of both the alpha(1B)-AR and alpha(1D)-AR inhibited DNA synthesis (as assessed by [(3)H]thymidine incorporation). In contrast, both receptors stimulated protein biosynthesis (as measured by [(35)S]methionine incorporation) and activated extracellular signal-regulated kinase (ERK)1/2. Importantly, these responses were agonist-dependent for the alpha(1B)-AR, but were agonist-independent for the alpha(1D)-AR. Agonist activation of the alpha(1B)-AR resulted in increased p38 kinase activity, but not c-Jun NH(2)-terminal kinase (JNK) activity, whereas the alpha(1D)-AR activated JNK but not p38 kinase. Unlike ERK1/2, JNK activity was increased by agonist treatment in the alpha(1D)-AR cells. An ERK1/2-pathway inhibitor PD98059 had no effect on phenylephrine-mediated inhibition of DNA synthesis in either cell line but blocked protein biosynthesis mediated by both receptors. The p38 kinase inhibitor SB203580 blocked alpha(1B)-AR effects on [(3)H]thymidine and [(35)S]methionine incorporation in alpha(1B)-AR-expressing cells, but had no effect on alpha(1D)-AR-mediated growth responses, consistent with the inability of the alpha(1D)-AR to activate p38 kinase. Therefore, alpha(1B)- and alpha(1D)-ARs mediated similar growth responses but differ with respect to the MAPK family member involved and the requirement for agonist.     

Heterodimerization with beta2-adrenergic receptors promotes surface expression and functional activity of alpha1D-adrenergic receptors

The alpha1D-adrenergic receptor (alpha1D-AR) is a G protein-coupled receptor (GPCR) that is poorly trafficked to the cell surface and largely nonfunctional when heterologously expressed by itself in a variety of cell types. We screened a library of approximately 30 other group I GPCRs in a quantitative luminometer assay for the ability to promote alpha1D-AR cell surface expression. Strikingly, these screens revealed only two receptors capable of inducing robust increases in the amount of alpha1D-AR at the cell surface: alpha1B-AR and beta2-AR. Confocal imaging confirmed that coexpression with beta2-AR resulted in translocation of alpha1D-AR from intracellular sites to the plasma membrane. Additionally, coimmunoprecipitation studies demonstrated that alpha1D-AR and beta2-AR specifically interact to form heterodimers when coexpressed in HEK-293 cells. Ligand binding studies revealed an increase in total alpha1D-AR binding sites upon coexpression with beta2-AR, but no apparent effect on the pharmacological properties of the receptors. In functional studies, coexpression with beta2-AR significantly enhanced the coupling of alpha1D-AR to norepinephrine-stimulated Ca2+ mobilization. Heterodimerization of beta2-AR with alpha1D-AR also conferred the ability of alpha1D-AR to cointernalize upon beta2-AR agonist stimulation, revealing a novel mechanism by which these different adrenergic receptor subtypes may regulate each other's activity. These findings demonstrate that the selective association of alpha1D-AR with other receptors is crucial for receptor surface expression and function and also shed light on a novel mechanism of cross talk between alpha1- and beta2-ARs that is mediated through heterodimerization and cross-internalization.     

The alpha(1A/C)- and alpha(1B)-adrenergic receptors are required for physiological cardiac hypertrophy in the double-knockout mouse

Catecholamines and alpha(1)-adrenergic receptors (alpha(1)-ARs) cause cardiac hypertrophy in cultured myocytes and transgenic mice, but heart size is normal in single KOs of the main alpha(1)-AR subtypes, alpha(1A/C) and alpha(1B). Here we tested whether alpha(1)-ARs are required for developmental cardiac hypertrophy by generating alpha(1A/C) and alpha(1B) double KO (ABKO) mice, which had no cardiac alpha(1)-AR binding. In male ABKO mice, heart growth after weaning was 40% less than in WT, and the smaller heart was due to smaller myocytes. Body and other organ weights were unchanged, indicating a specific effect on the heart. Blood pressure in ABKO mice was the same as in WT, showing that the smaller heart was not due to decreased load. Contractile function was normal by echocardiography in awake mice, but the smaller heart and a slower heart rate reduced cardiac output. alpha(1)-AR stimulation did not activate extracellular signal-regulated kinase (Erk) and downstream kinases in ABKO myocytes, and basal Erk activity was lower in the intact ABKO heart. In female ABKO mice, heart size was normal, even after ovariectomy. Male ABKO mice had reduced exercise capacity and increased mortality with pressure overload. Thus, alpha(1)-ARs in male mice are required for the physiological hypertrophy of normal postnatal cardiac development and for an adaptive response to cardiac stress.     

Pharmacological characterization and cross talk of alpha1a- and alpha1b-adrenoceptors coexpressed in human embryonic kidney 293 cells

We established three human embryonic kidney (HEK) 293 cell lines stably expressing alpha(1)-adrenoceptor (AR) subtypes, one (alpha(1A), (1B)-AR) coexpressing both receptors and the other two (alpha(1A)-AR and alpha(1B)-AR) expressing each receptor in isolation. In the alpha(1A), (1B)-AR cells, both receptors were clearly distinguished by the alpha(1A)-selective ligands (-)-1(3-hydroxypropyl)-5-((2R)-2-([2-(2,2,2-trifluoroethyl]oxy]phenyl)oxy)ethyl]amino)propyl)-2,3-dihydro-1H-indole-7-carboxamide (KMD-3213) and methoxamine, but not by the subtype-nonselective ligands prazosin and phenylephrine. In all three cell lines, phenylephrine caused a concentration-dependent increase in inositol phosphates and an increase in extracellular signal-regulated kinase 1/2 (ERK1/2) activation. However, there was a 2-fold or greater maximal response to phenylephrine and a somewhat higher agonist potency in ERK1/2 activation in the alpha(1A,1B)-AR cells, compared with the responses of cells expressing either receptor individually (alpha(1A)-AR or alpha(1B)-AR). Furthermore, the antagonistic affinities of prazosin (pK(b) of 10.1) and KMD-3213 (9.4) for inhibiting the phenylephrine response were intermediate between the values for inhibition in alpha(1A)-AR cells (prazosin, 9.3; KMD-3213, 10.5) and alpha(1B)-AR cells (prazosin, 11.0; KMD-3213, 8.1). The inhibitor pK(b) values in alpha(1A), (1B)-AR also differed from their ligand binding affinities measured in alpha(1A)-AR and alpha(1B)-AR cells. In contrast, the alpha(1A)-selective agonist methoxamine, which did not activate alpha(1B)-AR cells, stimulated either alpha(1A,) (1B)-AR or alpha(1A)-AR cells with a comparable potency and maximum effectiveness. Our data indicate that when coexpressed in the same cell, the activation of common pathways by individual AR receptor subtypes by a nonselective agonist can exhibit enhanced responsiveness and a distinct antagonist affinity compared with the parameters for the same receptors, when expressed alone in the same cell background.     

S18616, a highly potent, spiroimidazoline agonist at alpha(2)-adrenoceptors: I. Receptor profile, antinociceptive and hypothermic actions in comparison with dexmedetomidine and clonidine

S18616 ((S)-spiro[(1-oxa-2-amino-3-azacyclopent-2-ene)-4, 2'-(8'-chloro-1',2',3',4'-tetrahydronaphthalene)]) displayed high affinity at native rat alpha(2)-adrenoceptors (AR)s (pK(i), 9.8), native human (h)alpha(2A)-ARs (9.6), and cloned halpha(2A)- (9.5), halpha(2B)- (9.2), and halpha(2C)- (9.0) ARs. It showed 40-fold lower affinity for halpha(1A)-ARs (8.4) and >/=100-fold lower affinity for rat alpha(1)-ARs (7.1), halpha(1B)-ARs (7.7), halpha(1D)-ARs (7.6), imidazoline(1) (7.4), and imidazoline(2) (7.4) sites and >100-fold lower affinity for all other (>50) sites. At halpha(2A)-ARs, in guanosine-5'-O-(3-[(35)S]thio)triphosphate binding studies, S18616 was a potent (partial) agonist: log effective concentration (pEC(50)), 9.3/maximal effect, 51%. This observation was corroborated employing a halpha(2A)-Gi1alpha fusion protein/GTPase assay (9.0/40%) in which the actions of S18616 were blocked by pertussis toxin. Employing guanosine-5'-O-(3-[(35)S]thio)triphosphate binding assays, S18616 was also a partial agonist at halpha(2C)-ARs (8.2/63%) but a full agonist (8.4/124%) at halpha(2B)-ARs. At halpha(2A)-, halpha(2B)-, and halpha(2C)-ARs, the selective alpha(2)-AR antagonist, atipamezole, abolished the actions of S18616: pK(b) values of 9.1, 9. 1, and 9.4, respectively. As determined by depletion of membrane-bound [(3)H]phosphatidyl inositols, S18616 behaved as a (less potent) agonist (7.8/79%) at halpha(1A)-ARs, an action abolished by prazosin (pK(b), 8.9). Reflecting alpha(2)-AR agonist properties, S18616 potently (>/=1 microg/kg, s.c.) and dose dependently elicited hypothermia and antinociception (nine diverse models) in rodents. These actions were dose dependently inhibited by chemically diverse alpha(2)- versus alpha(1)-AR antagonists, atipamezole, idazoxan, RX821,002, and BRL44418 (a preferential alpha(2A)-AR ligand). In contrast, the actions of S18616 were unaffected by the alpha(1)-AR antagonists, ARC239 and prazosin (which preferentially block alpha(2B/2C)- versus alpha(2A)-ARs). Although the affinity of dexmedetomidine at alpha(2)-ARs was lower than S18616; it displayed a similar receptor and functional profile. Clonidine displayed lower efficacy than S18616, was substantially less potent, and had marked affinity for imidazoline(1) sites and alpha(1)-ARs. In conclusion, S18616 is a novel, selective, and highly potent agonist at alpha(2)-ARs.     

Pharmacological effects of ephedrine alkaloids on human alpha(1)- and alpha(2)-adrenergic receptor subtypes

Ephedra species of plants have both beneficial and adverse effects primarily associated with the presence of ephedrine alkaloids. Few reports have appeared that examine the direct actions of ephedrine alkaloids on human subtypes of adrenergic receptors (ARs). In the present study, ephedrine alkaloids were evaluated for their binding affinities on human alpha(1A)-, alpha(1B)-, alpha(1D)-, alpha(2A)-, alpha(2B)-, and alpha(2C)-AR subtypes expressed in HEK and Chinese hamster ovary cells. Cell-based reporter gene assays were used to establish functional activity of ephedrine alkaloids at alpha(1A)-, alpha(2A)-, and alpha(2C)-ARs. The data showed that ephedrine alkaloids did not activate alpha(1)- and alpha(2)-ARs and that they antagonized the agonist-mediated effects of phenylephrine and medetomidine on alpha(1)- and alpha(2)-ARs, respectively. As in the binding studies, 1R,2R- and 1R,2S-ephedrine showed greater functional antagonist activity than the 1S,2R- and 1S,2S-isomers. The rank order of affinity for the isomers was 1R,2R > 1R,2S > 1S,2R > 1S,2S. The rank order of potencies of alkaloids containing a 1R,2S-configuration was norephedrine > or = ephedrine > N-methylephedrine. These studies have demonstrated that orientation of the beta-hydroxyl group on the ethylamino side chain and the state of N-methyl substitution are important for alpha-AR binding and functional activity of the ephedrine alkaloids. In conclusion, the ephedrine isomers and analogs studied did not exhibit any direct agonist activity and were found to possess moderate antagonist activities on cloned human alpha-ARs. The blockade of presynaptic alpha(2A)- and alpha(2C)-ARs may have a pharmacological role in the direct actions of Ephedra alkaloids.     

Reduction of cardiac outward currents by alpha-1 adrenoceptor stimulation: a subtype-specific effect?

In rat ventricular myocytes, the effects of alpha adrenoceptor stimulation on outward currents were studied by means of the whole cell voltage-clamp technique. Phenylephrine (30 microM) in the presence of propranolol (1 microM) to block beta adrenoceptors reduced voltage-activated transient outward current. Both components of transient outward current were affected, i.e., peak current (Ipeak) was reduced by 25.3 +/- 1.8%, the outward current at the end of a clamp step (Ilate) was reduced by 39.1 +/- 3.5% (n = 5; holding potential -40 mV, clamp step to +20 mV). In order to describe the alpha-1 adrenoceptor subtypes involved in this action, the effect of phenylephrine was also investigated after pretreatment of the cells with various antagonists. Pretreatment with prazosin (0.3 microM) abolished completely the phenylephrine effect. The alpha-1A adrenoceptor subtype-selective antagonists 5-methylurapidil and (+)-niguldipine (0.1 microM each) and the irreversible alpha-1B adrenoceptor subtype antagonist chloroethyl-clonidine (100 microM) blocked the phenylephrine effect on Ipeak, but merely attenuated the effect on Ilate, whereas pretreatment with a combination of chloroethylclonidine and (+)-niguldipine suppressed the phenylephrine-induced effect on both outward current components just like prazosin did. In conclusion, stimulation of both adrenoceptor subtypes is required for reduction of Ipeak, but stimulation of either alpha-1A or alpha-1B subtype is sufficient for reduction of Ilate. Therefore, stimulation of both alpha-1 adrenoceptor subtypes contributes to the phenylephrine-induced reduction in transient outward currents of isolated rat myocytes.     

beta3-adrenergic receptor activation increases human atrial tissue contractility and stimulates the L-type Ca2+ current

beta3-adrenergic receptor (beta3-AR) activation produces a negative inotropic effect in human ventricles. Here we explored the role of beta3-AR in the human atrium. Unexpectedly, beta3-AR activation increased human atrial tissue contractility and stimulated the L-type Ca2+ channel current (I Ca,L) in isolated human atrial myocytes (HAMs). Right atrial tissue specimens were obtained from 57 patients undergoing heart surgery for congenital defects, coronary artery diseases, valve replacement, or heart transplantation. The I(Ca,L) and isometric contraction were recorded using a whole-cell patch-clamp technique and a mechanoelectrical force transducer. Two selective beta3-AR agonists, SR58611 and BRL37344, and a beta3-AR partial agonist, CGP12177, stimulated I(Ca,L) in HAMs with nanomolar potency and a 60%-90% efficacy compared with isoprenaline. The beta3-AR agonists also increased contractility but with a much lower efficacy (approximately 10%) than isoprenaline. The beta3-AR antagonist L-748,337, beta1-/beta2-AR antagonist nadolol, and beta1-/beta2-/beta3-AR antagonist bupranolol were used to confirm the involvement of beta3-ARs (and not beta1-/beta2-ARs) in these effects. The beta3-AR effects involved the cAMP/PKA pathway, since the PKA inhibitor H89 blocked I(Ca,L) stimulation and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) strongly increased the positive inotropic effect. Therefore, unlike in ventricular tissue, beta3-ARs are positively coupled to L-type Ca2+ channels and contractility in human atrial tissues through a cAMP-dependent pathway.     

Alpha1-adrenergic receptors augment P2X3 receptor-mediated nociceptive responses in the uninjured state.

In the present study, the adrenergic receptor (AR) subtype mediating adrenergic augmentation of P2X(3) receptor-mediated nociceptive responses on sensory nerve endings was examined by using selective AR receptor agonists and antagonists in Sprague Dawley rats in the uninjured state. Local administration of alphabeta-methyleneATP (ligand for P2X3/P2X2/3 receptors) into the plantar hind paw produced few pain behaviors when given alone in this strain of rats; combination with adrenaline (alpha1- and alpha2-AR agonist) and phenylephrine (alpha1-AR agonist) but not clonidine or UK 14,304 (alpha2-AR agonists) increased flinching behaviors. Flinching produced by noradrenaline (NA)/alphabeta-methyleneATP was suppressed by low doses of prazosin (alpha1-AR antagonist), and this reduction was selective compared with yohimbine (alpha2-AR antagonist). Prazosin also reduced flinching produced by phenylephrine/alphabeta-methyleneATP. Using thermal threshold determinations, adrenaline and phenylephrine but not clonidine or UK 14,304, mimicked the action of NA in augmenting reductions in thermal thresholds produced by alphabeta-methyleneATP. Terazosin (another alpha1-AR antagonist) inhibited hyperalgesia produced by NA/alphabeta-methyleneATP. These results provide evidence for alpha1-AR involvement in adrenergic augmentation of P2X3/P2X2/3 receptor-mediated responses on sensory nerve endings in the uninjured state in Sprague Dawley rats. PERSPECTIVE: This study indicates the alpha1-adrenergic receptor subtype mediates adrenergic augmentation of the activation of sensory nerves by purinergic P2X3 receptors (respond to ATP) in the periphery. Observations are potentially relevant to chronic pain conditions in which sympathetic nerves influence sensory nerves.     

Cardiac 7-transmembrane-spanning domain receptor portfolios: diversify, diversify, diversify

Enhanced signaling in myocytes by the G protein Gq has been implicated in cardiac hypertrophy and the transition to heart failure. alpha1-Adrenergic receptors (alpha1-ARs) are members of the 7-transmembrane-spanning domain (7-TM) receptor family and signal via interaction with Gq in the heart. The specific effects of a loss of alpha1-AR signaling in the heart are explored by O'Connell et al. in this issue of the JCI (see the related article beginning on page 1005). Paradoxically, gene ablation of the alpha 1A and alpha 1B subtypes in mice results in a maladaptive form of reactive cardiac hypertrophy from pressure overload, with a predisposition to heart failure. Thus signaling to the alpha1-AR (compared with signaling from other receptors such as angiotensin receptors, which also couple to Gq) appears to be specifically required for a normal hypertrophic response. This represents another example of how receptors that share common G proteins have diversified, developing unique signaling programs. These findings may have particular clinical relevance because of the widespread use of alpha1-AR antagonists in the treatment of hypertension and symptomatic prostate enlargement.