alpha2-AR Agonist Research Articles

Evidence that negative regulation of wakefulness in neonatal rats is an intrinsic function of the brain alpha2A-adrenergic receptors

Previously, it was proposed that sedative and anesthetic effects of alpha2-adrenergic receptor (alpha2-AR) agonists may be exerted via neuronal networks normally implicated in the regulation of wakefulness. The aim of this study was to evaluate the role of A subtype of alpha2-ARs in the development of drug-independent anesthetic state induced by hypothermia in newborn rats. Using short interfering RNA (siRNA) gene-targeting strategy, we found that down-regulation of the brainstem alpha2A-AR expression resulted in a delay in the onset of hypothermia-induced anesthesia assessed by loss of righting reflex. Involvement of the brain alpha2A-ARs in this delay was confirmed by inability of clonidine, a subtype-nonselective alpha2-AR agonist, to prolong duration of hypothermia-induced anesthesia in siRNA-treated animals, while significant prolongation of this anesthetic state by the alpha2A-AR agonist was observed in control pups. The data suggest that negative regulation of the animal's waking state is an intrinsic function of the brainstem alpha2A-ARs activated by exogenous agonists, as well as by endogenous noradrenaline, also.

Hypnotic and Hypotensive Responses to the Alpha2‐adrenergic Agonists Are Enhanced and Accompanied by Increased G Protein Coupling to the Alpha2AAR in Spinophilin Null Mice

We have previously identified spinophilin (SP) as a critical regulator of alpha2 adrenergic receptor trafficking and signaling both in vitro and in vivo. Compared to wild type mice, mice lacking SP expression are more sensitive to alpha2‐agonist‐elicited sedation. In the present study, we assessed cardiovascular responses and hypnotic effects elicited by systemic administration of an alpha2‐agonist, UK14,304, in SP null mice. Our data demonstrate that mice lacking SP expression display dramatically enhanced and prolonged hypotensive, bradycardic, and hypnotic responses to alpha2AAR stimulation. These changes in sensitivity to alpha2AR agonist occur independent of any changes in alpha2AAR density or intrinsic affinity for UK14,304 in the brain of SP null mice when compared to wild type controls. In contrast, the data indicate that coupling of the alpha2AAR to cognate G proteins is enhanced in SP null mice, based on changes in guanine nucleotide regulation of UK14,304 binding and evidence of a larger fraction of alpha2AAR in the high affinity state in preparations from SP null mice when compared to those from wild type mice. This suggests that an enhanced receptor/G protein coupling efficiency in the absence of SP expression contribute to an increased responsiveness of alpha2AARs in SP null mice, indicating that SP may exert ongoing regulation of receptor‐G protein coupling in vivo.

Desensitization of vascular response in vivo: contribution of genetic variation in the α2B-adrenergic receptor subtype

Vascular alpha2B-adrenergic receptors (alpha2B-ARs) mediate vasoconstriction and contribute to peripheral regulation of vascular tone. In vitro, a common 301-303 deletion in the alpha2B-AR gene, ADRA2B, results in loss of alpha2B-AR desensitization. We examined the hypothesis that ADRA2B del301-303 or other common ADRA2B variants alter vascular desensitization in vivo. We measured sensitivity to a highly selective alpha2-AR agonist, dexmedetomidine, (0.01-1000 ng/min) in the dorsal hand vein in 41 healthy individuals. To induce desensitization a dose of dexmedetomidine that resulted in submaximal constriction was infused for 180 min and dorsal hand vein responses measured. Desensitization was defined as the ratio between the area-under-the-effect curve for each individual's response and the hypothetical area-under-the-effect curve assuming that the initial response had been maintained for 180 min (ratio below 1 reflecting desensitization). The relationship between six ADRA2B variants (one promoter, three coding, and two in the 3' untranslated region ) with an allele frequency of more than 5% and desensitization was determined. Forty-one individuals (22 men, 21 whites, age 18-45 years) were studied. The ADRA2B 301-303 deletion allele (ins/del and del/del, n = 18) was associated with resistance to desensitization [1.01 (interquartile range 0.90-1.06)] as compared with ins/ins homozygous individuals (n = 23) [0.91 (interquartile range 0.73-0.99)], P = 0.026. In addition, the -98 GG, 1182 CC, and 1776 CC genotypes were associated with significantly less desensitization than GC or CC, and CA or AA genotypes, respectively. Common ADRA2B variants contribute to the interindividual variability in vascular desensitization to an alpha2-AR agonist in vivo.

α2-Adrenergic Receptors Attenuate Secretagogue-Induced Endocytosis and Promote Exocytosis of Intestinal NHE2 and NHE3

Adrenergic agonists, through activation of intestinal epithelial alpha2-adrenergic receptors (alpha2AR), inhibit electrolyte secretion and promote absorption. The mechanisms of action to promote basal Na(+) absorption and inhibit stimulated secretion are not understood completely. The effects of alpha2-agonists on Na(+) transport were studied in a cell line, Caco2-3B, derived from the Caco2 cell line engineered to permanently express human alpha2A-adrenergic receptors. Serosal, but not mucosal, addition of the alpha2AR agonist N-(2,6-dichlorophenyl)-4,5-dihydro-1H-imidazol-2-amine (clonidine) increased Caco2-3B apical (22)Na(+) uptake, an effect not seen in the Caco2 parent line that lacks alpha2AR expression. This effect was blocked by the alpha2AR antagonist 17alpha-yohmban-16alpha-carboxylic acid methyl ester (yohimbine). Increased Na(+) uptake was paralleled by increased apical surface abundance of the sodium/hydrogen exchangers NHE2 and NHE3. No changes in total cell NHE2 and NHE3 expression were observed. Clonidine also inhibited both cAMP and Ca(2+)-induced decreases in apical Na(+) uptake and apical membrane NHE2 and NHE3 endocytosis stimulated by these agents. alpha2AR actions were mediated via stimulation of phospholipase C, and metabolism of arachidonic acid by an epoxygenase activity followed epidermal growth factor release and activation of the epidermal growth factor receptor, resulting in phosphatidylinositol-3-kinase and Akt stimulation. In summary, activation of intestinal epithelial alpha2AR significantly blocks the inhibition of apical Na(+) transporters by cAMP- and Ca(2+)-mediated pathways and also directly increases apical sodium/hydrogen exchange activities. By both blocking electrolyte secretion and promoting absorption, alpha2-agonists could be potent antidiarrheal agents that could directly counteract the actions of toxigenic pathogens and other secretagogues causing secretory diarrhea.

Dexmedetomidine Increases Hippocampal Phosphorylated Extracellular Signal–regulated Protein Kinase 1 and 2 Content by an α2-Adrenoceptor–independent Mechanism

Dexmedetomidine is a potent and selective alpha2-adrenoceptor (alpha2AR) agonist that exhibits a broad pattern of actions, including sedation, analgesia, and neuroprotection. Recent studies have emphasized the role of phosphorylated extracellular signal-regulated protein kinases (pERK1 and 2) in coupling rapid events such as neurotransmitter release and receptor stimulation long-lasting changes in synaptic plasticity and cell survival. Here, the authors hypothesized that dexmedetomidine increases pERK1 and 2 content and examined the mechanisms involved in this effect. The effects of dexmedetomidine and their sensitivity to various pharmacologic agents on expression of pERK1 and 2 were studied by Western blots in hippocampal slices obtained from rats, wild-type mice, and mice carrying targeted deletions of the alpha2AR subtypes. Dexmedetomidine induced a concentration-related increase in the expression of pERK1 and 2 in rat hippocampal slices (EC50 [95% confidence interval] for pERK1, 0.97 [0.68-1.37] microm; for pERK2, 1.15 [0.62-2.14] microm). This effect was insensitive to the inhibitors of the alpha2AR-mediated signaling pathway, to prazosin, and to PP2, an inhibitor of the focal adhesion kinase-Src kinases. In contrast, it was still present in mice deleted for each of the alpha2AR subtypes and was markedly decreased by the antagonist of the I1-imidazoline receptors efaroxan, by phospholipase C and protein kinase C inhibitors, and by PD 098059, a direct inhibitor of ERK1 and 2 phosphorylation. Dexmedetomidine increases the expression of pERK1 and 2 via mechanisms independent of alpha2AR activation. The I1-imidazoline receptors likely contribute to these effects. The results may be relevant to some long-term effects (e.g., neuroprotective) of dexmedetomidine in the brain.

ST91 [2-(2,6-Diethylphenylamino)-2-imidazoline Hydrochloride]-Mediated Spinal Antinociception and Synergy with Opioids Persists in the Absence of Functional α-2A- or α-2C-Adrenergic Receptors

Agonists acting at alpha2-adrenergic receptors (alpha2ARs) produce antinociception and synergize with opioids. The alpha2ARs are divided into three subtypes, alpha(2A)AR, alpha(2B)AR, and alpha(2C)AR. Most alpha2AR agonists require alpha(2A)AR activation to produce antinociception and opioid synergy. The same subtype also mediates the side effect of sedation, which limits the clinical utility of these compounds. Identification of a non-alpha(2A)AR-mediated antinociceptive agent would enhance the therapeutic utility of alpha2AR agonists in pain management. Previous studies have suggested that the alpha2AR agonist ST91 [2-(2,6-diethylphenylamino)-2-imidazoline hydrochloride] has a nonsedating, non-alpha(2A)AR mechanism of action. We examined the pharmacology of spinal ST91 and its interaction with the delta-opioid agonist deltorphin II (Tyr-D-Ala-Phe-Glu-Val-Val-Gly amide) in mice lacking either functional alpha(2A)ARs or alpha(2C)ARs. All drugs were administered by direct lumbar puncture, and drug interactions were evaluated using isobolographic analysis. In contrast to the majority of alpha2AR agonists, ST91 potency was only moderately reduced (3-fold) in the absence of the alpha(2A)AR. Studies with the alpha2AR subtype-preferring antagonists BRL-44408 (2-[2H-(1-methyl-1,3-dihydroisoindole)methyl]-4,5-dihydroimidazole maleate) and prazosin [[4-(4-amino-6,7-dimethoxy-quinazolin-2-yl) piperazin-1-yl]-(2-furyl)methanone] and the pan-alpha2AR antagonist SKF-86466 (6-chloro-2,3,4,5-tetrahydro-3-methyl-1-H-3-benzazepine) suggest a shift from alpha(2A)AR to the other alpha2AR subtype(s) in the absence of alpha(2A)AR. Antinociceptive synergy with deltorphin II was preserved in the absence of either alpha(2A)AR or alpha(2C)AR. In conclusion, ST91 activates both alpha(2A)AR and non-alpha(2A)AR subtypes to produce spinal antinociception and opioid synergy. This study confirms that the spinal pharmacology of ST91 differs from that of other alpha2AR agonists and extends those data to include spinal synergy with opioid agonists. The unique profile of ST91 may be advantageous in pain management.

α1-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. 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.

In Vivo Endothelial Denudation Disrupts Smooth Muscle Caveolae and Differentially Impairs Agonist-Induced Constriction in Small Arteries

Experiments were performed to determine the effects of endothelial denudation in vivo on vasoconstrictor responses of mouse tail artery segments in vitro. A sterile wire (70 microm diameter) was inserted into tail arteries of anesthetized mice to mechanically denude the endothelium, and the animals were allowed to recover for 48 hours. The function of pressurized tail artery segments was then studied in vitro. Intimal injury markedly reduced endothelium-dependent relaxation to acetylcholine. Constriction evoked by the selective alpha1-adrenoceptor (alpha1-AR) agonist, phenylephrine, was not affected by in vivo endothelial denudation, indicating that the contractile function of vascular smooth muscle cells (VSMCs) was not impaired. However, constriction to the selective alpha2-AR agonist UK14304 or to endothelin-1 was significantly inhibited. Confocal microscopy of intact tail arteries localized caveolin-1 to punctuate structures, arranged in rows on or close to the surface of VSMCs. After in vivo endothelial denudation, this pattern was disrupted and caveolin-1 was localized to intracellular sites. When VSMC caveolae were disrupted in control arteries using the cholesterol acceptor methyl-beta-cyclodextrin, there was a similar impairment in constriction to endothelin-1 or alpha2-AR stimulation, but not alpha1-AR activation. These results suggest that intimal injury to small cutaneous arteries disrupts VSMC surface caveolae and selectively impairs constriction to stimuli that are dependent on these structures for signaling.

Direct Evidence for Imidazoline (I1) Receptor Modulation of Ethanol Action on Norepinephrine‐Containing Neurons in the Rostral Ventrolateral Medulla in Conscious Spontaneously Hypertensive Rats

Enhancement of the rostral ventrolateral medulla (RVLM) presympathetic (norepinephrine, NE) neuronal activity represents a neurochemical mechanism for the pressor effect of ethanol. In this study, we tested the hypothesis that ethanol action on RVLM presympathetic neurons is selectively influenced by the signaling of the local imidazoline (I1) receptor. To support a neuroanatomical and an I1-signaling selectivity of ethanol, and to circumvent the confounding effects of anesthesia, the dose-related neurochemical and blood pressure effects of ethanol were investigated in the presence of selective pharmacological interventions that cause reduction in the activity of RVLM or nucleus tractus solitarius (NTS) NE neurons via local activation of the I1 or the alpha2-adrenergic receptor in conscious spontaneously hypertensive rats. Local activation of the I1 receptor by rilmenidine (40 nmol) or by the I1/alpha2 receptor mixed agonist clonidine (1 nmol), and local activation of the alpha2-adrenergic receptor (alpha2AR) by the pure alpha2AR agonist alpha-methylnorepinephrine (alpha-MNE, 10 nmol) caused reductions in RVLM NE, and blood pressure. Intra-RVLM ethanol (1, 5, or 10 microg), microinjected at the nadir of the neurochemical and hypotensive responses, elicited dose-dependent increments in RVLM NE and blood pressure in the presence of local I1--but not alpha2-receptor activation. Only intra-NTS alpha-MNE, but not rilmenidine or clonidine, elicited reductions in local NE and blood pressure; ethanol failed to elicit any neurochemical or blood pressure responses in the presence of local activation of the alpha2AR within the NTS. The findings support the neuroanatomical selectivity of ethanol, and support the hypothesis that the neurochemical (RVLM NE), and the subsequent cardiovascular, effects of ethanol are selectively modulated by I1 receptor signaling in the RVLM.

Adrenal GRK2 upregulation mediates sympathetic overdrive in heart failure

Cardiac overstimulation by the sympathetic nervous system (SNS) is a salient characteristic of heart failure, reflected by elevated circulating levels of catecholamines. The success of beta-adrenergic receptor (betaAR) antagonists in heart failure argues for SNS hyperactivity being pathogenic; however, sympatholytic agents targeting alpha2AR-mediated catecholamine inhibition have been unsuccessful. By investigating adrenal adrenergic receptor signaling in heart failure models, we found molecular mechanisms to explain the failure of sympatholytic agents and discovered a new strategy to lower SNS activity. During heart failure, there is substantial alpha2AR dysregulation in the adrenal gland, triggered by increased expression and activity of G protein-coupled receptor kinase 2 (GRK2). Adrenal gland-specific GRK2 inhibition reversed alpha2AR dysregulation in heart failure, resulting in lowered plasma catecholamine levels, improved cardiac betaAR signaling and function, and increased sympatholytic efficacy of a alpha2AR agonist. This is the first demonstration, to our knowledge, of a molecular mechanism for SNS hyperactivity in heart failure, and our study identifies adrenal GRK2 activity as a new sympatholytic target.

Regulation of alpha2A‐AR trafficking by clonidine and guanfacine in native neurons

Despite intensive studies on alpha2-adrenergic receptor (AR) trafficking in heterologous systems, the properties of different agonists in inducing alpha2-AR internalization and subsequent sorting in native cells remain elusive. We have developed a mouse line in which N-terminal HA-tagged alpha2A-AR expression is driven by the endogenous mouse alpha2A-AR locus. This HA-alpha2A-AR knock-in mouse line allows us to study alpha2A-AR trafficking studies in native cells. Two alpha2-AR agonists, clonidine and guanfacine have been widely used to treat attention deficit and hyperactivity disorder clinically, however, these two drugs can cause different degree of sedative response on patients. In mice, we found that the EC50 of clonidine to induce sedation is 20 folds higher than that of guanfacine. To understand the molecular and cellular mechanisms underlying selective regulation of alpha2A-AR-mediated physiological/pharmacological responses by these two agonists, we studied alpha2A-AR trafficking and ERK signaling in native neocortical neurons induced by these agonists, exploiting the HA-alpha2A-AR knock-in mice. We found that alpha2A-AR internalization induced by clonidine occurred more rapidly than that induced by guanfacine. Our studies provide the first documentation of endogenously expressed alpha2A-AR trafficking in native neocortical neurons. This research is supported by American Heart Association Scientist Development Grant to QW.

Mixed β3‐adrenoceptor agonist and α1‐adrenoceptor antagonist properties of nebivolol in rat thoracic aorta

Nebivolol, a selective beta-adrenoceptor (beta1-AR) antagonist, induces vasodilatation by an endothelium- and NO-cGMP-dependent pathway. However, the mechanisms involved in the vascular effect of nebivolol have not been established. Thus, we evaluated the role of alpha1 and beta3-ARs in nebivolol-induced vasodilatation. The responses to nebivolol were investigated in vitro in thoracic aortic rings isolated from male Sprague-Dawley rats. Nebivolol (0.1-10 microM) significantly shifted the concentration-response curve to phenylephrine, an alpha1-AR agonist, to the right in a concentration-dependent manner (pA2 = 6.5). Conversely, the concentration-response curve to endothelin 1 (ET1) was unaffected by nebivolol. In ET1-precontracted rings, nebivolol induced a concentration-dependent relaxation, which was unaffected by nadolol (a beta1/beta2-AR antagonist) but was significantly reduced by L-748,337 (a beta3-AR antagonist), endothelium removal or pretreatment with L-NMMA (an NOS inhibitor). Similar results were obtained with a beta3-AR agonist, SR 58611A. It was concluded that, in rat aorta, nebivolol-induced relaxation results from both inhibition of alpha1-ARs and activation of beta3-ARs. In addition, we confirmed that the endothelium and the NO pathway are involved in the vascular effect of nebivolol. The identification of these vascular targets of nebivolol indicate that it has therapeutic potential for the treatment of pathological conditions associated with an elevation of sympathetic tone, such as heart failure and hypertension.

Role of metalloproteinase‐dependent EGF receptor activation in α1‐adrenoceptor‐stimulated MAP kinase phosphorylation in GT1‐7 neurons

Adrenoceptors (ARs) are involved in the regulation of gonadotropin-releasing hormone (GnRH) release from native and immortalized hypothalamic (GT1-7) neurons. However, the AR-mediated signaling mechanisms and their functional significance in these cells are not known. Stimulation of GT1-7 cells with the alpha1-AR agonist, phenylephrine (Phe), causes phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) mitogen-activated protein (MAP) kinases that is mediated by protein kinase C (PKC)-dependent transactivation of the epidermal growth factor receptor (EGF-R). Phe stimulation causes shedding of the soluble ligand, heparin-binding EGF (HB-EGF), as a consequence of matrix metalloproteinase (MMP) activation. Phe-induced phosphorylation of the EGF-R, and subsequently of Shc and ERK1/2, was attenuated by inhibition of MMP or HB-EGF with the selective inhibitor, CRM197, or by a neutralizing antibody. In contrast, phosphorylation of the EGF-R, Shc and ERK1/2 by EGF and HB-EGF was independent of PKC and MMP activity. Moreover, inhibition of Src attenuated ERK1/2 responses by Phe, but not by HB-EGF and EGF, indicating that Src acts upstream of the EGF-R. Consistent with a potential role of reactive oxygen species (ROS), Phe-induced phosphorylation of EGF-R was attenuated by the antioxidant, N-acetylcysteine. These data suggest that activation of the alpha1-AR causes phosphorylation of ERK1/2 through activation of PKC, ROS and Src, and shedding of HB-EGF, which binds to and activates the EGF-R.

The selective alpha-adrenergic receptor agonist dexmedetomidine (DEX) does not affect aortic pressure augmentation

Background Despite a similar reduction in peripheral blood pressure (BP), antihypertensive drugs have variable effects on central blood pressure and its augmentation by the reflected pulse wave, a measure of arterial stiffness. Infusion of norepinephrine (NE) increases arterial stiffness. Centrally acting alpha2-AR agonists decrease NE concentrations and blood pressure, but their effects on aortic pressure augmentation are not known. Methods In a single-blind, placebo-controlled study, 36 healthy subjects received sequential infusions of placebo and DEX (0.1, 0.15 and 0.15 mcg/kg). Brachial artery blood pressure, heart rate, and NE plasma concentrations were measured at baseline and after every infusion. Radial artery waveforms were recorded by applanation tonometry (Sphygmocor) and the central aortic waveform and augmentation index - the proportional increase in systolic pressure due to the reflected wave - calculated. Results DEX reduced brachial and central systolic (−15.0±6.2 (SD) and −12.6±5.8 mmHg) and diastolic BP (−9.9±6.5 and −10.1±6.6 mmHg), respectively (p<0.001). NE levels decreased (−103±68 pg/mL, p<0.001) but augmentation index did not change (placebo 3.4±10.3% vs DEX 3.0±10.1%, P=0.8). Conclusion DEX reduced NE concentrations and central and peripheral blood pressure but did not alter aortic pressure augmentation. Pharmacological reduction of endogenous baseline NE concentrations did not alter arterial stiffness and pulse wave reflection. Clinical Pharmacology & Therapeutics (2005) 77, P11–P11; doi: 10.1016/j.clpt.2004.11.046

??2B Adrenergic receptor 301???303 deletion polymorphism and vascular ??2 adrenergic receptor response

Postsynaptic alpha2B adrenergic receptors (ARs) mediate vasoconstriction. There is more than 1000-fold variability in vascular sensitivity to an alpha2-AR agonist. Genetic variability may contribute to such interindividual differences in sensitivity. A 301-303 deletion (del) polymorphism has been identified in the coding region of the alpha2B-AR gene and has functional effects in vitro. Thus, we examined the hypothesis that the del301-303 polymorphism contributes to variability in vascular alpha2-AR responses in vivo. Healthy subjects were recruited based on their alpha2B-AR genotype. Their vascular sensitivity was determined using a linear variable differential transformer following the infusion of increasing doses (range 0.01-1000 ng/min) of the alpha2-AR agonist, dexmedetomidine, into a dorsal hand vein. The dose that produced 50% (ED50) of maximum venoconstriction (Emax) was determined for each subject. Vascular response was compared among the three genotypes. Forty-nine subjects were studied [28 wild-type wt/wt, 13 wt/del, 8 del/del]. There was no difference in dexmedetomidine ED50 and Emax among the alpha2B-AR del301-303 genotypes. The ED50 was 1.39 ng/min [95% confidence interval (CI) 0.03-63.0 ng/min] in wt/wt subjects, 1.63 ng/min (95% CI 0.01-177.8 ng/min) in wt/del and 2.37 ng/min (95% CI 0.17-33.7 ng/min) in del/del (P=0.80). The average Emax was 75.4+/-14.9% in wt/wt, 75.7+/-21.3% in wt/del and 82.2+/-12.9% in del/del subjects (P=0.26). These findings suggest that the del301-303 polymorphism does not contribute significantly to interindividual in vivo variability in response to alpha2-AR activation in the hand vein.

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.

EFFECTS OF α 1-ADRENERGIC RECEPTOR SUBTYPE SELECTIVE ANTAGONISTS ON LOWER URINARY TRACT FUNCTION IN RATS WITH BLADDER OUTLET OBSTRUCTION

Antagonists of alpha 1-adrenergic receptors (alpha 1ARs) relieve obstructive and irritative symptoms in patients with bladder outlet obstruction. However, to our knowledge mechanisms underlying the relief of irritative symptoms remain unknown. Because bladder alpha 1dARs are up-regulated in some rats with bladder outlet obstruction, we investigated the effect of the alpha 1aAR antagonist 5-methyl urapidil (5MU) vs the alpha 1a/alpha 1dAR antagonist tamsulosin on urinary frequency in obstructed rats. Baseline frequency was measured using a chronic micturition recording system and then obstruction (40 rats) or sham obstruction surgery (11 rats) was performed. After 6 weeks frequency was reassessed, followed by subcutaneous implantation of osmotic pumps to deliver 5MU, tamsulosin or vehicle for 1 week. Upon the completion of drug treatment urinary frequency was again measured and the pressor response to the alpha 1AR agonist phenylephrine was documented. Obstructed bladder mass was an average of 4.9 times greater than bladder mass in sham operated rats (p <0.001). Urinary frequency was elevated in obstructed rats with a bladder mass of greater than 500 mg vs all rats with a bladder mass of under 255 mg (p = 0.01). Of rats with a bladder mass of greater than 500 mg frequency was decreased in those treated with tamsulosin (p = 0.03) but not in those treated with 5MU. Tamsulosin and 5MU inhibited the pressor response to phenylephrine. Urinary frequency is increased in rats with a bladder mass of greater than 500 mg. The combined alpha 1a/alpha 1dAR antagonist tamsulosin decreases urinary frequency more than the alpha 1aAR selective antagonist 5MU. This finding supports the hypothesis that the alpha 1dAR is important for mediating irritative symptoms.

Adrenergic regulation of the intracellular [Ca2+] and voltage-operated Ca2+ channel currents in the rat prostate neuroendocrine cells.

The prostate gland contains numerous neuroendocrine cells (PNECs) innervated by adrenergic neurons. PNECs are believed to influence the growth and physiological function of the prostate gland via paracrine release of hormones. Using fura-2 fluorescence measurement and patch-clamp techniques, we investigated the effects of adrenergic stimulation on cytosolic concentration of Ca2+ ([Ca2+]c) and high voltage-activated Ca2+ channel currents (HVA-I(Ca)) of the putative rat prostate neuroendocrine cells (RPNECs) freshly isolated by an enzymic digestion. Noradrenaline (NA, 1 microM) induced a sharp, transient increase of [Ca2+]c measured by the fura-2 fluorescence. Pharmacological studies showed that alpha1-adrenoceptors (alpha1-ARs) coupled with PLC/IP3 signaling pathway induce the release of stored Ca2+, which subsequently recruits store-operated Ca2+ entry pathways. In the whole-cell voltage clamp experiment, NA decreased the amplitude of HVA-I(Ca) by 40%, which was mimicked by an alpha2-AR agonist (UK14304) but not by an alpha1-AR agonist (phenyleprine). After selective blockade of N-type Ca2+ channels by omega-conotoxin GVIA, the addition of NA showed no further inhibition on the remaining L-type Ca2+ channel currents. The adrenergic inhibition of HVA-I(Ca) was partially prevented by the pretreatment with pertussis toxin (PTX) (5 microg/ml, 4 hr, 37 degrees C). RPNECs express both alpha1- and alpha2-ARs, signaling the release of stored Ca2+ and the inhibition of N-type Ca2+ channels, respectively.

Mechanism of the negative inotropic effects of alpha 1-adrenoceptor agonists on mouse myocardium.

This study was done to identify the mechanism of the alpha1-adrenoceptor (AR) mediated negative inotropic effects of phenylephrine (PE) on adult mouse myocardium. As reported by others, we also found that the nonselective alpha1AR agonist PE produced a negative inotropic effect on ventricular strips from adult mice that was inhibited by the alpha1AAR antagonist 5-methylurapidil (5MU) but not by the alpha1BAR antagonist chloroethylclonidine (CEC) or the alpha1DAR antagonist BMY 7378. The selective alpha1AAR agonist A61603 also produced a negative inotropic effect, which was antagonized by 5MU. Phorbol 12,13-dibutyrate (activator of all PKC isoforms) mimicked the negative inotropic responses to PE and A61603. The negative inotropic effects of PE were inhibited by bisindolylmaleimide (inhibitor of all PKC isoforms) but not by Gö 6976 (inhibitor of Ca2+-dependent PKC). Rottlerin, an inhibitor of Ca2+-independent PKCdelta, antagonized the negative inotropic effects of PE and A61603. PE and A61603 increased the translocation of PKCdelta, which was prevented by rottlerin. These data suggest that the alpha1AR-mediated negative inotropy on adult mouse myocardium is signaled by Ca2+-independent PKCdelta.

The role of I(1)-imidazoline and alpha(2)-adrenergic receptors in the modulation of glucose metabolism in the spontaneously hypertensive obese rat model of metabolic syndrome X.

We examined glucose metabolism after I1-imidazoline (I1R) and alpha2-adrenergic receptor (alpha2AR) activation in an animal model of metabolic syndrome X. Fasted spontaneously hypertensive obese rats (SHROB) were given the I1R/alpha2AR agonists moxonidine and rilmenidine or the alpha2AR agonist guanabenz. Because of the dual specificity of moxonidine, its actions were split into adrenergic and nonadrenergic components by using selective antagonists: rauwolscine (alpha2AR) efaroxan (I1R/alpha2AR), or 2-endo-amino-3-exo-isopropylbicyclo[2.2.1.]heptane (AGN 192403) (I1R). Hyperglycemia induced by moxonidine, rilmenidine, and guanabenz resulted from inhibition of insulin secretion. Similar responses were observed after oral dosing and in lean littermates. Glucagon was reduced by the I1R agonists (moxonidine, 32 +/- 5%; rilmenidine, 24 +/- 7%) but elevated by guanabenz (71 +/- 32%). The hyperglycemic and hypoinsulinemic responses to moxonidine were blocked by rauwolscine. In contrast, rauwolscine potentiated the reduction in glucagon (39 +/- 6%). AGN 193402 blocked the glucagon response without affecting hyperglycemia and hypoinsulinemia. Efaroxan blocked all responses to moxonidine. When SHROB rats were treated with moxonidine 15 min before an oral glucose tolerance test, the glucose area under the curve (AUC) was increased. Antagonizing the alpha2AR component of moxonidine's action with rauwolscine improved glucose AUC 3-fold and facilitated the insulin secretory response and reduced glucagon secretion. Testing fasting glucose and insulin during 3 weeks of oral moxonidine revealed early hyperglycemia that later faded, and a progressive drop in fasting insulin. The acute hyperglycemia and hypoinsulinemia elicited by moxonidine and rilmenidine was mediated by alpha2AR, whereas I1R may reduce glucagon and increase insulin, particularly after a glucose load.