alpha2-AR Agonist Research Articles

Exercise restores beta-adrenergic vasorelaxation in aged rat carotid arteries.

Aging is associated with alterations in beta-adrenergic receptor (beta-AR) signaling and reduction in cardiovascular responses to beta-AR stimulation. Because exercise can attenuate age-related impairment in myocardial beta-AR signaling and function, we tested whether training could also exert favorable effects on vascular beta-AR responses. We evaluated common carotid artery responsiveness in isolated vessel ring preparations from 8 aged male Wistar-Kyoto (WKY) rats trained for 6 wk in a 5 days/wk swimming protocol, 10 untrained age-matched rats, and 10 young WKY rats. Vessels were preconstricted with phenylephrine (10-6 M), and vasodilation was assessed in response to the beta-AR agonist isoproterenol (10-10-3 x 10-8 M), the alpha2-AR agonist UK-14304 (10-9-10-6 M), the muscarinic receptor agonist ACh (10-9-10-6 M), and nitroprusside (10-8-10-5 M). beta-AR density and cytoplasmic beta-AR kinase (beta-ARK) activity were tested on pooled carotid arteries. beta-ARK expression was assessed in two endothelial cell lines from bovine aorta and aorta isolated from a 12-wk WKY rat. beta-AR, alpha2-AR, and muscarinic responses, but not that to nitroprusside, were depressed in untrained aged vs. young animals. Exercise training restored beta-AR and muscarinic responses but did not affect vasodilation induced by UK-14304 and nitroprusside. Aged carotid arteries showed reduced beta-AR number and increased beta-ARK activity. Training counterbalanced these phenomena and restored beta-AR density and beta-ARK activity to levels observed in young rat carotids. Our data indicate that age impairs beta-AR vasorelaxation in rat carotid arteries through beta-AR downregulation and desensitization. Exercise restores this response and reverts age-related modification in beta-ARs and beta-ARK. Our data support an important role for beta-ARK in vascular beta-AR vasorelaxation.

Regulation of Cl- secretion by alpha2-adrenergic receptors in mouse colonic epithelium.

Previous studies have shown that alpha2 adrenoceptor (alpha2AR) agonists inhibit electrolyte secretion in colonic epithelia, but little is known about the molecular mechanisms involved in this process. In this study we examined the effect of alpha2AR activation on transepithelial anion secretion across isolated murine colonic epithelium. We found that alpha2AR agonists, UK 14,304, clonidine and medetomidine were potent inhibitors of anion secretion, especially in the proximal colon. Short circuit current measurements (Isc) in colonic epithelia from normal and cystic fibrosis (CF) mice showed that alpha2AR agonists inhibited basal cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl- secretion but had no effect on CFTR activation by cAMP-dependent phosphorylation. Apical administration of an ionophore, nystatin (90 microg ml-1), was used to investigate the effect of UK 14,304 on basolateral K+ transport. The Na+-K+-ATPase current, measured as ouabain-sensitive current in the absence of ion gradients, was unaltered by pretreatment of the tissue with UK 14,304 (1 microM). In the presence of a basolaterally directed K+ gradient, UK 14,304 significantly reduced nystatin-activated Isc indicating that activation of alpha2ARs inhibits basolateral K+ channels. Studies with selective K+ channel inhibitors and openers showed that alpha2AR agonists inhibited KATP channels that were tonically active in mouse colonic epithelia. RT-PCR and pharmacological studies suggested that these channels could be similar to vascular smooth muscle KATP channels comprising Kir6.1/SUR2B or Kir6.2/SUR2B subunits. Inhibition of anion secretion by alpha2AR agonists required activation of pertussis toxin-sensitive Gi/o proteins, but did not involve classical second messengers, such as cAMP or Ca2+. In summary, alpha2ARs inhibit anion secretion in colonic epithelia by acting on basolateral KATP channels, through a process that does not involve classical second messengers.

Mutation of the α2A-Adrenoceptor Impairs Working Memory Performance and Annuls Cognitive Enhancement by Guanfacine

Norepinephrine strengthens the working memory, behavioral inhibition, and attentional functions of the prefrontal cortex through actions at postsynaptic alpha2-adrenoceptors (alpha2-AR). The alpha2-AR agonist guanfacine enhances prefrontal cortical functions in rats, monkeys, and human beings and ameliorates prefrontal cortical deficits in patients with attention deficit hyperactivity disorder. The present study examined the subtype of alpha2-AR underlying these beneficial effects. Because there are no selective alpha2A-AR, alpha2B-AR, or alpha2C-AR agonists or antagonists, genetically altered mice were used to identify the molecular target of the action of guanfacine. Mice with a point mutation of the alpha2A-AR, which serves as a functional knock-out, were compared with wild-type animals and with previously published studies of alpha2C-AR knock-out mice (Tanila et al., 1999). Mice were adapted to handling on a T maze and trained on either a spatial delayed alternation task that is sensitive to prefrontal cortical damage or a spatial discrimination control task with similar motor and motivational demands but no dependence on prefrontal cortex. The effects of guanfacine on performance of the delayed alternation task were assessed in additional groups of wild-type versus alpha2A-AR mutant mice. We observed that functional loss of the alpha2A-AR subtype, unlike knock-out of the alpha2C-AR subtype, weakened performance of the prefrontal cortical task without affecting learning and resulted in loss of the beneficial response to guanfacine. These data demonstrate the importance of alpha2A-AR subtype stimulation for the cognitive functions of the prefrontal cortex and identify the molecular substrate for guanfacine and novel therapeutic interventions.

Subtype-specific neuronal differentiation of PC12 cells transfected with α 2-adrenergic receptors

Cells of the PC12 rat pheochromocytoma cell line acquire characteristics of sympathetic neurons under appropriate treatment. Stably transfected PC12 cells expressing individual alpha2-adrenergic receptor (alpha2-AR) subtypes were used to assess the role of alpha2-ARs in neuronal differentiation and to characterise the signalling pathways activated by the alpha2-AR agonist epinephrine in these cells. The effects of alpha2-AR activation were compared with the differentiating action and the signalling mechanisms of nerve growth factor (NGF). Epinephrine induced neuronal differentiation of PC12alpha2 cells through alpha2-AR activation in a subtype-dependent manner, internalization of all human alpha2-AR subtypes, and activation of mitogen-activated protein kinase (MAPK) and the serine-threonine protein kinase Akt. Epinephrine and NGF showed synergism in their differentiating effects. The MAPK kinase (MEK-1) inhibitor PD 98059 abolished the differentiating effect of epinephrine indicating that the differentiation is dependent on MAPK activation. Activating protein-1 (AP-1) DNA-binding activity was increased after epinephrine treatment in all three PC12alpha2 subtype clones. Evaluation of the potential physiological consequences of these findings requires further studies on endogenously expressed alpha2-ARs in neuronal cells.

Modification of ureteral motility and promotion of urine flow around an intraureteral obstruction by CL‐316243, phenylephrine, and furosemide in dogs

The aim of this study was to evaluate the effects of a beta3-adrenoceptor (AR) agonist (CL-316243), an alpha1-AR agonist (phenylephrine), and a loop diuretic (furosemide) on the spontaneous rhythmic contractions of the isolated canine ureter and on an acute ureteral obstruction produced by inflation of a balloon catheter in anesthetized dogs. In the isolated ureter, CL-316243 concentration dependently reduced both the amplitude and frequency of the rhythmic contractions (pD(2): 7.19 +/- 0.33), whereas phenylephrine significantly enhanced both variables (pD(2): 5.26 +/- 0.09) and furosemide reduced them only slightly. In the acute ureteral obstruction model, the intraureteral pressure (IUP) gradually rose to reach a plateau of 58.9 mm Hg after inflation of a balloon catheter within the lower ureter. Intravenous administration of CL-316243 (0.3 microg/kg) significantly reduced the elevated IUP and the resumed urine flow (UF), leading to a sustained reduction in the IUP. In contrast, the IUP continued to increase above the plateau level for 10 minutes after phenylephrine administration (10 microg/kg) and for 30 minutes after furosemide administration (1,000 microg/kg). In the phenylephrine group, the UF resumed when the IUP reached 75.8 mm Hg, and thereafter the IUP gradually decreased in parallel with the increase in the UF. From these results, we conclude that in dogs, CL-316243 reduces the IUP by allowing the UF to resume as a result of a relaxation of ureter at the obstruction site, whereas with phenylephrine, the reduction in the IUP is secondary to a resumption in the UF resulting from an induced contraction of ureter that causes an increase in hydrostatic pressure above the obstruction site.

Effects of testosterone on alpha2A-adrenergic receptor expression in the rat brain.

Androgens are involved in regulation of behaviour through intracellular mechanisms owing to their receptors. Involvement of intercellular messengers such as brain norepinephrine and adrenergic receptors (ARs) is seemed to be necessary to realise hormone-dependent behavioural effects. Castration of adult male rats, which decreases copulatory activity in the animals, was accompanied by a significant increase in 3H-clonidine (alpha2-AR agonist) binding site density in the frontal cortex. The levels of mRNA for the alpha2A-ARs (measured by RT-PCR) were increased in the brainstem of castrated males in parallel to the changes in cortical ARs densities. Testosterone treatment, that activates copulatory behaviour in castrates, down regulated alpha2A-AR mRNA levels in the brainstem and 3H-clonidine binding sites densities in the cortex, where terminals of the brain stem neurones are situated. Unlike in the brainstem, castration caused a decrease in alpha2A-AR mRNA in the cortex and testosterone up-regulated this mRNA in the cortical region. The data suggested that down-regulation of alpha2-ARs densities in the cortex that is induced by testosterone can be preferentially related to alpha2-ARs subpopulation which is expressed by the brainstem neurones and imported into the cortex by axons of these neurones.

Cooling Evokes Redistribution of α2C-Adrenoceptors from Golgi to Plasma Membrane in Transfected Human Embryonic Kidney 293 Cells

Cold-induced vasoconstriction in cutaneous blood vessels is mediated by increased constrictor activity of vascular alpha2-adrenoceptors (alpha2-ARs). In mouse cutaneous arteries, alpha2-AR constriction at 37 degrees C is mediated by alpha2A-ARs, whereas after cold exposure (28 degrees C), alpha2C-ARs are no longer silent and mediate the remarkable cold-induced augmentation of alpha2-AR responsiveness. The goals of the present study were to develop a cell model of cutaneous thermoregulation and to determine the mechanisms underlying the thermosensitivity of alpha2C-ARs. Human embryonic kidney 293 cells were transiently transfected with the mouse alpha2A- or alpha2C-AR. In cells expressing alpha2A-ARs, UK-14,304 (5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine), an alpha2-AR agonist, inhibited (10 pM) and stimulated (1-10 nM) the accumulation of cAMP evoked by forskolin. Similar responses were obtained at 37 degrees C and 28 degrees C. In contrast, in cells expressing alpha2C-ARs, UK-14,304 did not affect forskolin-stimulated cAMP accumulation at 37 degrees C but did cause a concentration-dependent inhibitory effect at 28 degrees C. Subcellular fractionation revealed that at 37 degrees C alpha2C-ARs were localized predominantly to Golgi compartments, whereas alpha2A-ARs localized predominantly to the plasma membrane. After cooling (28 degrees C), alpha2C-ARs relocated from Golgi compartments to the plasma membrane, whereas the alpha2A-AR remained at the plasma membrane. Immunofluorescence microscopy confirmed that, at 37 degrees C, alpha2A-ARs were localized to the cell surface, whereas alpha2C-ARs colocalized with a trans-Golgi marker. Cooling did not affect localization of alpha2A-ARs, but shifted alpha2C-ARs to the cell surface. Moderate cooling, therefore, caused a selective redistribution of alpha2C-ARs from the Golgi compartments to the cell surface, allowing the rescue of the alpha2C-adrenergic functional response. This mechanism may explain the role of alpha2-ARs in thermoregulation of the cutaneous circulation.

The alpha2 -adrenergic receptors in hypertension and heart failure: experimental and clinical studies.

This is a brief overview of experimental and clinical studies exploring the hemodynamic functions of the alpha2A and alpha2B adrenergic receptor (AR) subtypes in animals submitted to genetic manipulations or gene treatment, as well as the clinical effects of central sympathetic suppression with the alpha2-AR agonist clonidine in patients with ischemic heart disease and/or heart failure. The animal experiments have led us to conclude that the sympathetic outflow is regulated by activation of the presynaptic alpha2A-AR subtype, which is the predominant alpha2-AR subtype in the central nervous system and exerts a sympathoinhibitory (hypotensive) action; on the contrary, activation of the central alpha2B-AR elicits a sympathoexcitatory response (such as seen in salt-induced hypertension, which requires functionally intact alpha2B-AR). Since there are no selective pharmacologic agents yet capable of discriminating among alpha2-AR subtypes, clinical studies utilize clonidine, the central sympathetic suppressant effect of which has been used for 35 years to treat hypertension. In small clinical trials, clonidine was used successfully for treatment of acute or chronic heart failure, acute myocardial infarct or hypertensive cardiomyopathy with subclinical diastolic dysfunction. We speculate that future development of agents capable of selectively activating the alpha2A-AR or blocking the alpha2B-AR may further improve our capability to treat hypertension, ischemic heart disease and heart failure.

Stimulation of alpha1-adrenoceptors inhibits memory consolidation in the chick.

Investigation of the effects of the different adrenoceptor (AR) subtypes in memory formation may reveal discrete actions of noradrenaline in memory modulation and storage mediated through particular AR subtypes. Noradrenaline injected intracerebrally in the chick produced biphasic effects on memory consolidation with enhancement at low doses and inhibition at high doses. We have previously shown that the enhancement by the lower doses of noradrenaline is attributable to actions at beta2- and beta3-adrenoceptors, whereas the inhibitory effect of higher doses is attributable to alpha1-adrenoceptors. The present studies show that the inhibition of memory by high doses of noradrenaline is mimicked by the alpha1-AR agonist methoxamine, and the dose-response curve is shifted to the right by pretreatment with the alpha1-AR antagonist prazosin. alpha1-ARs may play a critical role in memory formation in highly stressful situations, when noradrenaline levels are high in particular brain regions. It is not known where the alpha1-ARs responsible for the effect on memory are localized. alpha1-ARs are found on neurons and astrocytes and in the cerebral vasculature and therefore the action of high doses of noradrenaline via alpha1-AR agonists could be via an action at any of these sites. Activation of alpha1-adrenoceptors in the intermediate hyperstriatum ventrale in the chick forebrain by the alpha1 adrenoceptor agonist methoxamine inhibits the consolidation of memory. Because the same effect is produced by high levels of noradrenaline, it is likely that stimulation of alpha1-ARs is the mechanism underlying this effect.

Effects of central imidazolinergic and alpha2-adrenergic activation on water intake.

Non-adrenergic ligands that bind to imidazoline receptors (I-R), a selective ligand that binds to alpha2-adrenoceptors (alpha2-AR) and mixed ligands that bind to both receptors were tested for their action on water intake behavior of 24-h water-deprived rats. All drugs were injected into the third cerebral ventricle. Except for agmatine (80 nmol), mixed ligands binding to I-R/alpha2-AR such as guanabenz (40 nmol) and UK 14304 (20 nmol) inhibited water intake by 65% and up to 95%, respectively. The selective non-imidazoline alpha2-AR agonist, alpha-methylnoradrenaline, produced inhibition of water intake similar to that obtained with guanabenz, but at higher doses (80 nmol). The non-adrenergic I-R ligands histamine (160 nmol, mixed histaminergic and imidazoline ligand) and imidazole-4-acetic acid (80 nmol, imidazoline ligand) did not alter water intake. The results show that selective, non-imidazoline alpha2-AR activation suppresses water intake, and suggest that the action on imidazoline sites by non-adrenergic ligands is not sufficient to inhibit water intake.

Cloning and characterization of the mouse alpha1C/A-adrenergic receptor gene and analysis of an alpha1C promoter in cardiac myocytes: role of an MCAT element that binds transcriptional enhancer factor-1 (TEF-1).

alpha1-Adrenergic receptor (AR) subtypes in the heart are expressed by myocytes but not by fibroblasts, a feature that distinguishes alpha1-ARs from beta-ARs. Here we studied myocyte-specific expression of alpha1-ARs, focusing on the subtype alpha1C (also called alpha1A), a subtype implicated in cardiac hypertrophic signaling in rat models. We first cloned the mouse alpha1C-AR gene, which consisted of two exons with an 18 kb intron, similar to the alpha1B-AR gene. The receptor coding sequence was >90% homologous to that of rat and human. alpha1C-AR transcription in mouse heart was initiated from a single Inr consensus sequence at -588 from the ATG; this and a putative polyadenylation sequence 8.5 kb 3' could account for the predominant 11 kb alpha1C mRNA in mouse heart. A 5'-nontranscribed fragment of 4.4 kb was active as a promoter in cardiac myocytes but not in fibroblasts. Promoter activity in myocytes required a single muscle CAT (MCAT) element, and this MCAT bound in vitro to recombinant and endogenous transcriptional enhancer factor-1. Thus, alpha1C-AR transcription in cardiac myocytes shares MCAT dependence with other cardiac-specific genes, including the alpha- and beta-myosin heavy chains, skeletal alpha-actin, and brain natriuretic peptide. However, the mouse alpha1C gene was not transcribed in the neonatal heart and was not activated by alpha1-AR and other hypertrophic agonists in rat myocytes, and thus differed from other MCAT-dependent genes and the rat alpha1C gene.

Analysis of ligand activation of alpha 2-adrenoceptor subtypes under conditions of equal G alpha protein stoichiometry.

Variations in the measurement of ligand's intrinsic activity between receptor subtypes is a common consequence of unequal receptor:G protein density ratios. We have investigated ligand activation at the alpha2-adrenoceptor (alpha2-AR) subtypes under defined expression conditions of one receptor molecule for one Galpha protein molecule using fusion proteins. Fusion between either a wt alpha2C AR or a mutant Thr382Lys alpha2C AR and a chimeric Galphaq/il protein displayed robust, transient (-)-adrenaline-mediated Ca2+ responses with similar potencies (pEC50: 7.78 and 7.66) and kinetic properties. A comparison of the intrinsic activities of alpha2 AR agonists found d-medetomidine to be the only compound with an efficacy similar to that of (-)-adrenaline. The Ca2+ responses as mediated by UK 14304, oxymetazoline and clonidine became more potent and efficacious at the Thr381Lys alpha2C AR, whereas the response as mediated by talipexole displayed a higher potency with an unaltered maximal response. Whereas only small differences in ligand's intrinsic activities between the wt alpha2A, alpha2B and alpha2C AR fusion proteins were observed with most ligands, oxymetazoline was virtually silent at the alpha2A AR while active as a partial and apparently full agonist at the alpha2C AR and alpha2B AR, respectively. The mutant alpha2 AR subtypes could be differentiated using the apparent positive efficacy of ligands that used to be defined as antagonists. The following rank order of maximal responses was observed for the Thr381Lys alpha2C AR: idazoxan approximately equals SKF 86466 > atipamezole >> dexefaroxan; Thr373Lys alpha2A AR: SKF 86466 > idazoxan = atipamezole > dexefaroxan; and Thr370Lys alpha2B AR: atipamezole > idazoxan dexefaroxan. RX 811059 (10 microM) was the only compound to be completely silent at both the wt and mutant alpha2 AR subtypes. In conclusion, silent alpha2 AR ligands are probably rare in these specified alpha2 AR systems. Most antagonists may actually possess partial agonist properties at the alpha2 AR subtypes, which are facilitated by the same mutation in the distal portion of their third intracellular loop.

Effects of micromolar concentrations of manganese, copper, and zinc on alpha1-adrenoceptor-mediating contraction in rat aorta.

To determine the influences of the Mn, Cu, and Zn on alpha1-adrenoceptor (AR)-mediated vasoconstriction, we investigated their effects on vasoconstriction produced by the alpha1-AR agonist phenylephrine in isolated rings of rat thoracic aorta. The cumulative concentration-contraction curves for phenylephrine were obtained in the absence and presence of Mn (0.3, 1, 3 microM), Cu (1, 10, 16 microM), and Zn (0.3, 1, 10 microM). Mn, Cu, and Zn each inhibited phenylephrine-mediated contraction in a dose-dependent manner. The maximal phenylephrine-induced contraction was significantly reduced by the pretreatment of the arterial rings with 10 and 16 microM Cu (p<0.05). The results suggest that variations in the plasma concentrations of metal might lead to changes in alpha1-AR-mediated constrictive response.

Increased alpha2-adrenergic constriction of isolated arterioles in diffuse scleroderma.

Vasospasm and ischemic organ injury are important in the pathogenesis of systemic sclerosis (SSc; scleroderma). The present study was performed to determine whether SSc arterioles have an intrinsic disturbance in vasoconstrictor activity. Skin biopsy samples were obtained from the upper arm of 11 patients with diffuse SSc (clinically uninvolved skin) and 8 age- and sex-matched control subjects. Dermal arterioles were dissected from the biopsy sample and mounted in a myograph for continuous monitoring of arteriolar diameter. The resting internal diameter of control and SSc arterioles was similar (mean +/- SEM 164+/-15 micro and 166+/-18micro, respectively). Dermal arterioles displayed no spontaneous constrictor activity in the absence of stimulation. Vasoconstriction in response to KCI, a receptor-independent activator of smooth muscle, or to phenylephrine, a selective alpha1-adrenergic receptor (alpha1-AR) agonist, was similar in control and SSc arterioles. However, constrictor responses to UK 14,304, a selective alpha2-AR agonist, were increased in SSc compared with control arterioles (maximal constriction responses of 25+/-5% and 67+/-4% [mean +/- SEM] in control and SSc arterioles, respectively; P = 0.000014). Mechanical denudation of the endothelium did not alter reactivity to alpha2-AR activation, indicating that the enhanced constriction in SSc was not mediated by changes in endothelial dilator activity. Indeed, in arterioles constricted with phenylephrine, the endothelial stimuli acetylcholine or bradykinin evoked endothelium-dependent relaxation that was similar in control and SSc arterioles. Vascular smooth muscle in SSc arterioles displayed a selective increase in alpha2-AR reactivity. The endothelial dilator function appeared normal. Altered activity of smooth muscle alpha2-ARs may contribute to the vasospastic activity that is a prominent feature of the SSc disease process.

Pregnancy switches adrenergic signal transduction in rat and human uterine myocytes as probed by BKCa channel activity

1. We used large conductance Ca2+-activated K+ (BKCa) channel activity as a probe to characterize the inhibitory/stimulatory G protein (Gi/Gs) signalling pathways in intact cells from pregnant (PM) and non-pregnant (NPM) myometrium. 2. Isoprenaline (10 microM) enhanced the outward current (Iout) in PM cells and inhibited Iout in NPM cells. Additional application of the alpha2-adrenoceptor (alpha2-AR) agonist clonidine (10 microM) further enhanced the isoprenaline-modulated Iout in PM cells but partially antagonized Iout in NPM cells. Clonidine alone did not affect Iout. The specific cAMP kinase (PKA) inhibitor H-89 (1 microM) abolished the effects of isoprenaline and clonidine. The specific BKCa channel blocker iberiotoxin (0.1 microM) inhibited Iout by approximately 80 %; the residual current was insensitive to isoprenaline. 3. Inhibition of Gi activity by either pertussis toxin or the GTPase activating protein RGS16 abolished inhibitory as well as stimulatory effects of clonidine on Iout. 4. Transducin-alpha, a scavenger of Gi betagamma dimers, converted the stimulatory action of clonidine on Iout into an inhibitory effect. Free transducin-betagamma enhanced both the stimulatory and the inhibitory effects of isoprenaline on Iout. 5. The results demonstrate that BKCa channel activity is a sensitive probe to follow adenylyl cyclase-cAMP-PKA signalling in myometrial smooth muscle cells. Both Gialpha-mediated inhibition and Gibetagamma-mediated stimulation can occur in the same cell, irrespective of pregnancy. It is speculated that the coupling between alpha2-AR and Gi proteins is more efficient during pregnancy and that Gibetagamma at high levels simply override the inhibitory action of Gi alpha.

α2-Adrenergic Receptors in Human Dorsal Root Ganglia

Nonselective alpha2-adrenergic receptor (alpha2AR) agonists (e.g., clonidine) mediate antinociception in part through alpha2ARs in spinal cord dorsal horn; however, use of these agents for analgesia in humans is limited by unwanted sedation and hypotension. The authors previously demonstrated alpha2a approximately alpha2b > > > alpha2c mRNA in human spinal cord dorsal horn cell bodies. However, because 20% of dorsal horn alpha2ARs derive from cell bodies that reside in the associated dorsal root ganglion (DRG), it is important to evaluate alpha2AR expression in this tissue as well. Therefore, the authors evaluated the hypothesis that alpha2b mRNA, alpha2c mRNA, or both are present in human DRG. Molecular approaches were used to determine alpha2AR expression in 28 human DRGs because of low overall receptor mRNA expression and small sample size. After creation of synthetic competitor cDNA and establishment of amplification conditions with parallel efficiencies, competitive reverse transcription polymerase chain reaction was performed using RNA isolated from human DRG. Overall expression of alpha2AR mRNA in DRG is low but reproducible at all spinal levels. alpha2b and alpha2cAR subtype mRNAs predominate (alpha2b approximately alpha2c), accounting for more than 95% of the total alpha2AR mRNA in DRG at all human spinal nerve root levels. Predominance of alpha2b and alpha2cAR mRNA in human DRG is distinct from alpha2AR mRNA expression in cell bodies originating in human spinal cord dorsal horn, where alpha2a and alpha2b predominate with little or absent alpha2c expression. These findings also highlight species heterogeneity in alpha2AR expression in DRG. If confirmed at a protein level, these findings provide an additional step in unraveling mechanisms involved in complex neural pathways such as those for pain.

Cell signaling in bovine ciliary epithelial organ culture.

The ciliary epithelium secretes aqueous humor, an intraocular fluid whose production is regulated in part by transmembrane signaling pathways including those mediated by G protein-coupled receptors. Many drugs, such as beta-adrenergic receptor (AR) antagonists and alpha2-AR agonists, are used to lower intraocular pressure by presumably decreasing fluid transport across this epithelium. Hence, our purpose was to establish a ciliary epithelial organ culture system suitable for the study of cell signaling pathways. A trypsin-mediated dissection method was established to isolate bovine ciliary epithelial sheets. These sheets were cultured in a 5% CO2 incubator. The quality was assessed by light microscopy, by protein analysis, and by the evaluation of epinephrine-mediated phosphoinositide turnover. The cultured epithelial explants were viable as evidenced by minimal trypan blue staining. The explants were composed primarily of nonpigmented cells and some pigmented cells, but no other ciliary body tissues were present on histology. Membrane preparations showed proteins with a distribution from 31 to 116 kDa. Epinephrine caused a dose-dependent increase in [3H]inositol phosphates (InsPs) accumulation with a maximal increase of two- to three-fold over basal levels. This epinephrine-mediated increase was inhibited by prazosin. We established an organ culture system of isolated bovine ciliary epithelium suitable for the study of transmembrane signaling pathways.

Catecholamines and angiotensinogen gene expression in kidney proximal tubular cells.

To investigate the molecular mechanism(s) of action of catecholamines on the expression of the angiotensinogen (ANG) gene in kidney proximal tubular cells, we used opossum kidney (OK) cells with a fusion gene containing the 5'-flanking regulatory sequence of the rat ANG gene fused with a human growth hormone (hGH) gene as a reporter, pOGH (rANG N-1498/+18), permanently integrated into their genomes. The level of expression of the ANG-GH fusion gene was quantified by the amount of immunoreactive-hGH (IR-hGH) secreted into the medium. The addition of norepinephrine (NE), isoproterenol (a beta1/beta2-adrenergic receptor (AR) agonist) and iodoclonidine (an alpha2-AR agonist) stimulated the expression of the ANG-GH fusion gene in a dose-dependent manner, whereas the addition of epinephrine and phenylephrine (alpha1-AR agonist) had no effect. The stimulatory effect of NE was blocked by the presence of propranolol (beta-AR blocker), atenolol (beta1-AR blocker), yohimbine (alpha2-AR blocker), Rp-cAMP (an inhibitor of cAMP-dependent protein kinase AI & AII) and staurosporine (an inhibitor of protein kinase C), but was not blocked by ICI 118, 551 (beta2-AR blocker) and prazosin (alpha1-AR blocker). The addition of a combination of isoproterenol and iodoclonidine or a combination of 8-Bromo-cAMP (8-Br-cAMP) and phorbol 12-myristate (PMA) synergistically stimulated the expression of the ANG-GH fusion gene as compared to the addition of isoproterenol, iodoclonidine, 8-Br-cAMP or PMA alone. Furthermore, the addition of NE, 8-Br-cAMP or PMA stimulated the expression of pOGH (rANG N-806/-779/-53/+18), a fusion gene containing the putative cAMP responsive element (CRE, ANG N-806/-779) upstream of the ANG promoter (ANG N-53/+18) in OK cells, but had no effect on the expression of fusion genes containing the mutant of the CRE. Gel mobility shift assays revealed that the ANG-CRE binds with the DNA-binding domain (bZIP254-327) of the cAMP-responsive binding protein (CREB). The binding of the labeled ANG-CRE to CREB (bZIP254-327) was displaced by unlabeled ANG-CRE and the CRE of the somatostatin gene but not by the mutants of the ANG-CRE. Finally, NE stimulated the phosphorylation of CREB in OK cells. These studies demonstrate that the molecular mechanism(s) of NE action on the expression of the ANG gene in OK cells may be mediated via both the PKA and PKC signalling pathways and via the phosphorylation of CREB. The phosphorylated CREB then interacts with the CRE in the 5'-flanking region of the ANG gene and subsequently stimulates the gene expression.

Effects of alpha1-adrenoceptor antagonists on cultured prostatic smooth muscle cells.

alpha1-adrenoceptor (alpha1-AR) antagonists, used to relieve the lower tract urinary symptoms (LUTS) in benign prostate hyperplasia (BPH) patients, are thought to act in inhibiting the contraction of stromal smooth muscle. An attempt was made using new technology to visualize and quantify the effect of alpha1-AR antagonists in a cell culture model of prostatic smooth muscle cells (SMC). Prostatic smooth muscle cells cultured from human prostate tissue were treated with alpha1-AR agonists and antagonists. The effects on cell growth, cell contraction, differentiation status, and apoptosis were determined by means of an MTT cell viability assay, time-lapse video microscopy, RT-PCR analysis, and FACS analysis of annexin V/propidium iodide-stained cells, respectively. Prostatic smooth muscle cells derived from prostate tissue expressed SMC-specific markers. They showed spontaneous contractions, and phenylephrine increased the percentage of contracting cells by 3-fold. alpha1-AR antagonists inhibited spontaneous as well as phenylephrine-induced contractions. Long-term treatment with doxazosin induced differentiation tended towards a contractile phenotype, as indicated by an increase of the ratio of smooth muscle heavy chain myosin subtypes SM2/SM1. There was, however, no effect on cell growth. High concentrations of antagonist (100 microM) induced apoptosis in about 80% of the treated SMC. This effect was not cell-type-specific and was also seen in skin fibroblasts and immortalized prostate epithelial cells. In an easy-to-handle cell culture model of prostatic smooth muscle cells, the effects of alpha1-AR antagonists on cell contraction, growth, and differentiation can be investigated. The results indicate that in addition to inhibition of cell contraction, alpha1-AR antagonists have the potential to induce apoptosis.

Cardiovascular α&lt;sub&gt;1&lt;/sub&gt;-adrenoceptor subtypes: functions and signaling

alpha-Adrenoceptors (alpha1AR) are G protein-coupled receptors and include alpha1A, alpha1B, and alpha1D subtypes corresponding to cloned alpha1a, alpha1b, and alpha1d, respectively. alpha1AR mediate several cardiovascular actions of sympathomimetic amines such as vasoconstriction and cardiac inotropy, hypertrophy, metabolism, and remodeling. alpha1AR subtypes are products of separate genes and differ in structure, G protein-coupling, tissue distribution, signaling, regulation, and functions. Both alpha(1A)AR and alpha(1B)AR mediate positive inotropic responses. On the other hand, cardiac hypertrophy is primarily mediated by alpha(1A)AR. The only demonstrated major function of alpha(1D)AR is vasoconstriction. alpha1AR are coupled to phospholipase C, phospholipase D, and phospholipase A2; they increase intracellular Ca2+ and myofibrillar sensitivity to Ca2+ and cause translocation of specific phosphokinase C isoforms to the particulate fraction. Cardiac hypertrophic responses to alpha1AR agonists might involve activation of phosphokinase C and mitogen-activated protein kinase via Gq x alpha1AR subtypes might interact with each other and with other receptors and signaling mechanisms.