Rauwolscine Binding Research Articles

In vivo stimulation of the beta(2)-adrenergic pathway increases expression of the Gi proteins and the alpha(2)A-adrenergic receptor genes in the pregnant rat myometrium.

Cross-regulations between Gs and Gi mediated pathways controlling the adenylyl cyclase activity have been clearly demonstrated in vitro. To elucidate whether activation of the beta-adrenergic pathway in the pregnant myometrium might affect Gi proteins and alpha(2)-adrenergic receptors (ARs), we treated late pregnant rats from day 18 to day 21 with twice-daily administration of isoproterenol (8 mg/kg). This treatment increased myometrial cAMP levels and led after 76 h to a significant and maximal rise in the immunoreactive amount of myometrial Gi alpha 2 and Gi alpha 3 proteins (1.4- and 1.7-fold respectively) associated with a parallel increase of the steady-state levels of both Gi alpha 2 and Gi alpha 3 mRNA (1.6- and 1.9-fold respectively). Propranolol antagonized this response indicating the implication of the beta-adrenergic pathway. Nuclear run-on assays demonstrated that isoproterenol enhanced respectively by 1.3- and 1.2-fold the transcription rate of the Gi alpha 2 and Gi alpha 3 genes. Quantification of myometrial alpha(2)-ARs by [3H]rauwolscine binding revealed that the total number of receptors was also increased at 76 h by 1.7-fold when compared with controls, with no change in the affinity of the alpha(2)-ARs for the ligand. This effect was antagonized by propranolol. Quantification of both alpha(2A)- and alpha(2B)-subtypes by Northern blotting analysis demonstrated that this elevation was due to a selective increase of the alpha(2A)-subtype mRNAs. The present results indicate that in vivo stimulation of the beta-adrenergic pathway by isoproterenol increases both Gi alpha 2/Gi alpha 3 and alpha(2A)-AR expression in the pregnant rat myometrium. The possible contribution of such a mechanism in pregnancy-related changes of both entities is discussed.

Heterogeneity of alpha 2-adrenoceptors in human and rat myometrium and differential expression during pregnancy.

1. The aim of this study was first, to characterize alpha 2-adrenoceptor subtypes in human and rat pregnant myometrium and second, to investigate the possibility of a differential expression of the putative subtypes according to the stage of pregnancy. 2. In both species, specific [3H]-rauwolscine binding was inhibited by five different compounds with an order of affinity characteristic of the one described for alpha 2-adrenoceptors (yohimbine > or = clonidine > noradrenaline > phenylephrine > propranolol). Binding affinities (pKi) for the compounds tested were, in human and rat, respectively: 7.63 and 8.93 for yohimbine, 6.91 and 8.71 for clonidine, 6.23 and 6.09 for noradrenaline, 5.37 and 5.73 for phenylephrine, 4.64 and 4.72 for propranolol. 3. By use of non-linear iterative curve fitting procedures and by fitting the data to a two-site model, analysis of [3H]-rauwolscine inhibition binding curves performed in the presence of oxymetazoline (alpha 2A-selective), ARC239, prazosin or chlorpromazine (alpha 2B- and alpha 2C-selective) indicated that pregnant human and rat myometrium contain at least two pharmacologically distinct alpha 2-adrenoceptor subtypes (alpha 2A, alpha 2B and/or alpha 2C). RNA blot analysis with probes specific for each cloned human and rat alpha 2-adrenoceptor subtype demonstrated that alpha 2A- and alpha 2B-subtypes were present in both species but alpha 2C seems to be expressed only in human tissues. 4. In the pregnant rat myometrium, subtype selective compounds competition curves revealed a predominant expression of alpha 2A-adrenoceptors at mid-pregnancy whereas, at term, alpha 2A- and alpha 2B-subtypes density reached approximately the same level (alpha 2A:alpha 2B ratio = 73:27 at mid-pregnancy and = 43:57 at term). In addition, quantification of alpha 2A- and alpha 2B-transcripts by densitometry, following data normalization with an oligo(dT)12-18 probe, showed a pattern of expression comparable to the one characterized by pharmacological studies. 5. In conclusion, these data demonstrate heterogeneity of alpha 2-adrenoceptors in pregnant human and rat myometria and an alteration of the alpha 2A-/alpha 2B-subtypes expression pattern during rat pregnancy. Such observations lead us to suggest a multiple role for alpha 2-adrenoceptors in regulating specific functions of myometrium throughout the time course of pregnancy.

Characterization of alpha-adrenoceptors in canine mesenteric vein.

The alpha-adrenergic receptors (alpha-ARs) in canine mesenteric vein (DMV) were studied by using nonselective agonists and selective antagonists in functional studies and in ligand binding to classify the subtypes present. Based on functional studies of phenylephrine (PE)-induced contractions and ligand-binding interactions of [3H]-prazosin with prazosin (PR), WB 4101 (WB), 5-methylurapidil (5-MU), BMY 7378, and SK&F 105854, and pretreatment with chloroethylclonidine (CEC), DMV alpha1-ARs resembled the alpha1D subtype. However, the affinity of PR assessed in functional and ligand-binding studies was less (pK(B,D,i) < or = 9) than expected from previous characterization of cloned rat or human alpha1-AR (pKi > or = 10). Interactions with 5-MU, BMY 7378, or SK&F 105854 suggested the presence of some alpha1-ARs that were not typical of alpha1D-AR and that binding and functional interactions did not yield corresponding results. PR binding was abolished by treatment with CEC, contractile responses to PE were reduced in Emax, and the concentration-effect curve shifted to the left, as previously reported. DMVs contracted in response to alpha2-AR agonists and were studied when contractions were potentiated by increasing extracellular KCl to 20 mM. Rauwolscine (RAU) had K(B) values at these sites consistent with K(D) values in binding studies. CEC had no effect on RAU binding in DMV. Ligand-binding studies to [3H]-RAU sites did not reveal a clear identification of subtype, but these alpha2-ARs were clearly not alpha2B-ARs. We conclude that canine mesenteric vein contains alpha1D-like ARs, but with significant differences, and an unclassifiable alpha2-AR. There may also be a smaller population of other, not alpha1D-like ARs, receptors, mediating responses to PE and binding of prazosin.

Cloning and Expression of a Complementary DNA Encoding a Molluscan Octopamine Receptor That Couples to Chloride Channels in HEK293 Cells

A cDNA encoding a G-protein-coupled receptor was cloned from the central nervous system of the pond snail Lymnaea stagnalis. The predicted amino acid sequence of this cDNA most closely resembles the Drosophila tyramine/octopamine receptor, the Locusta tyramine receptor, and an octopamine receptor (Lym oa1) that we recently cloned from Lymnaea. After stable expression of the cDNA in HEK293 cells, we found that [3H]rauwolscine binds with high affinity to the receptor (KD = 6.2.10(-9) M). Octopamine appears to be the most potent naturally occurring agonist to displace the [3H]rauwolscine binding (Ki = 3.0.10(-7) M). Therefore, the receptor is considered to be an octopamine receptor and is consequently designated Lym oa2. The novel receptor shares little pharmacological resemblance with Lym oa1, indicating that the two receptors represent different octopamine receptor subfamilies. Octopaminergic stimulation of Lym oa2 does not induce changes in intracellular concentrations of cAMP or inositol phosphates. However, electrophysiological experiments indicate that octopamine is able to activate a voltage-independent Cl- current in HEK293 cells stably expressing Lym oa2. Although opening of this chloride channel most probably does not require the activation of either protein kinase A or C, it can be blocked by inhibition of protein phosphorylation.

Expression of α2 adrenoceptors during rat brain development—II. α2C messenger RNA expression and [ 3H]rauwolscine binding

The distributions of α 2C adrenoceptor messenger RNA and high-affinity [ 3H]rauwolscine binding sites were characterized in developing rat brain. Using in situ hybridization with 35S-labeled riboprobes directed against the third intracellular loop, α 2C messenger RNA expression appeared in an adult-like pattern during the first and second postnatal weeks, in the anterior olfactory nucleus, caudate–putamen, olfactory tubercles, islands of Calleja and hippocampus, following the time-course of maturation of these structures. Only in the cerebellum was α 2C messenger RNA transiently expressed during the critical period of granule cell development. High-affinity [ 3H]rauwolscine binding sites were detected using receptor autoradiography and revealed a similar spatial and temporal time-course of appearance during rat brain development. The highest numbers of binding sites were detected in the olfactory tubercles and islands of Calleja, and moderate numbers in the anterior olfactory nucleus, caudate–putamen and hippocampus. Like α 2C messenger RNA expression, high-affinity [ 3H]rauwolscine binding sites were transiently expressed in the cerebellum. In some areas (e.g., the substantia nigra), [ 3H]rauwolscine binding sites were detected even though α C2 messenger RNA expression was absent. The strong spatial and temporal correspondence between messenger RNA expression and radioligand binding supports the conclusion that [ 3H]rauwolscine selectively labels α 2C adrenoceptors in the rat brain. The developmental pattern which was observed is in marked contrast to the early, transient expression of the α 2A adrenoceptor. Thus, the α 2A and α 2C receptor types may serve distinct functional roles in the developing brain.

α2 A/ D-Adrenoceptor subtype predominates also in the neonatal rat spinal cord

α 2-Adrenoceptors are remarkably regulated by developmental factors. In this study α 2-adrenoceptor subtypes have been characterised in neonatal and adult rat spinal cords. In saturation experiments, a 5% proportion of [ 3H]rauwolscine binding has a high affinity component, representing the α 2C-subtype in both tissues. Competition studies with [ 3H]RX821002 indicate that in both tissues the α 2 A/ D subtype is expressed similarly.

Polyclonal anti-idiotypic antibodies to idazoxan and their interaction with human brain imidazoline binding sites

Polyclonal antibodies were raised in rabbits against purified polyclonal anti-idazoxan antibodies. The anti-idiotypic antibodies thus obtained, proved able to inhibit [ 3H]idazoxan specific binding to anti-idazoxan antibodies. Applied to human nucleus reticularis lateralis membrane preparations, these antibodies (20 μg) inhibited about 50 and 70% of the imidazoline specific binding of [ 3H]idazoxan and [ 3H]clonidine, respectively. Furthermore, they specifically immunoprecipitated 50% of [ 3H]idazoxan binding activity of imidazoline binding sites solubilized from the same tissue. [ 3H]Rauwolscine binding to α 2-adrenoceptors in rat cortex was not significantly affected by these antibodies. The antibodies labeled a 43 kDa protein in Western blots of partially purified imidazoline binding sites from human brain. In conclusion, these anti-idiotypic antibodies recognize imidazoline binding sites from human brain and allow the detection of a 43 kDa binding protein associated with or representing the imidazoline receptor expressed in human brain.

Unusual αadrenoceptor subtype in canine saphenous vein: comparison to mesenteric vein

1. We investigated the nature of the adrenoceptors in the dog saphenous vein (DSV) and dog mesenteric vein (DMV) to determine the nature of the unexpected interactions of phenylephrine and methoxamine with rauwolscine in the DSV, i.e. the ability of the putative alpha 2-adrenoceptor antagonist to inhibit competitively contractions to these alpha 1-agonists. Radioligand binding studies were performed in parallel with contractility studies. 2. Functionally, in the DSV, phenylephrine and methoxamine-induced, contractions were antagonized by rauwolscine with Schild slopes of -0.52 and -0.46, respectively and apparent pA2 values of 8.5 and 9.2, respectively. Such antagonism was not observed in the DMV. In the DSV, prazosin competes for [3H]-rauwolscine binding sites with a high and a low affinity binding site (Ki of 1.49 +/- 0.65 and 94.7 +/- 51 microM, n = 6, respectively). 3. Pretreatment with 100 microM chloroethylclonidine (CEC) for 15 min abolished [3H]-prazosin binding in microsomes from both veins and reduced binding (Bmax) of [3H]-rauwolscine in microsomes by 55.1 +/- 0.8% (n = 3) in the DSV but did not affect the Bmax in the DMV. CEC pretreatment in the venular rings denuded of endothelium caused persistent contraction in the DSV but not in the DMV. In the DSV, CEC appeared to interact with a single [3H]-rauwolscine binding site. In both the DSV and the DMV, CEC (100 microM) caused a significant shift in the EC50 values for phenylephrine and methoxamine. Maximum responses in the DMV were significantly attenuated while those in the DSV were unaffected when total tension was considered. 4. Studies of the functional interactions of the DSV and the DMV with WB 4101 or 5-methylurapidil (5-MU) suggested the presence of alpha 1D-adrenoceptors in the DSV and alpha 1A-adrenoceptors in the DMV. The receptors inactivated by CEC in the DMV and DSV may represent some or all of the receptors with properties of alpha 1D and alpha 1A-receptors present in the two veins. Studies of radioligand binding interactions of these two antagonists with [3H]-prazosin, were consistent with the presence of some alpha 1D-receptors in DSV and alpha 1A-receptors in DMV. These findings raise questions about the selectivity of CEC in differentiating alpha 1-adrenoceptor subtypes. 5. B-HT 920 caused contractions in the DSV smaller than those to the alpha 1-agonists but the maximum was not affected by CEC pretreatment. The EC50 values were shifted to the left after CEC. In radioligand binding studies, B-HT 920 competition for [3H]-rauwolscine binding was not significantly affected by CEC pretreatment. 6. These results suggest the presence of unusual alpha-adrenoceptors in the DSV. In addition to alpha 2-adrenoceptors, receptors recognizing rauwolscine as well as prazosin, WB 4101, phenylephrine and methoxamine and susceptible to inactivation by CEC are present. They appear to be, in part, unusual alpha 1D-adrenoceptors.

A‐80426, a potent α2‐adrenoceptor antagonist with serotonin uptake blocking activity and putative antidepressant‐like effects: I. Biochemical profile

AbstractA‐80426 (N‐[2‐(benzofuran‐6‐yl)ethyl]‐N‐[(R)−( + )‐5‐methoxy‐1,2,3,4‐tetrahydronaphthalen‐1‐yl methyl]‐N‐methylamine) is a novel compound with potential antidepressant activity. A‐80426 inhibits synaptosomal serotonin (5HT) uptake (IC50 = 13 nM) more potently than fluoxetine (IC50 = 308 nM), and blocks [3H]‐paroxetine binding (K1 = 3.8 nM) to 5HT uptake sites. A‐80426 inhibits [3H]‐rauwolscine binding to α2‐adrenoceptors (K1 = 2.0 nM), blocks α2‐adrenoceptors (pEC30 = 7.4–7.5) in electrically stimulated rat atria and vas deferens, and increases electrically stimulated [3H]‐NE overflow from brain slices. A‐80426 lacks significant activity at isolated tissue models of α1‐ or β‐adrenergic sites or at M3 muscarinic or H1 histaminergic receptors. A‐80426 has negligible radioligand binding activity at 5HT1 and β‐adrenergic receptors, but exhibits a K1 value of 144 nM at 5HT2 receptors. A‐80426 interacts with both dopamine D1 (K1 = 744 nM) and D2 (K1 = 51.5 nM) receptors. Functionally, the compound antagonizes dopamine‐induced changes in cyclic AMP formation with K1 values of 133 nM and 185 nM at D1 and D2 receptors, respectively. The potent α2‐adrenoceptor blocking activity of A‐80426 in radioligand binding assays is not reflected in isolated tissue bioassays, and may thus have less functional consequence in determining the in vivo profile of the compound. Accompanying behavioral data [Giardina et al., 1995], while indicating activity in the olfactory bulbectomized rat model of antidepressant action, shows a marked reduction in the α2‐antagonistic properties of A‐80426 in other in vivo models, suggesting that the antidepressant‐like activity may be caused primarily by inhibition of 5HT uptake. © 1995 Wiley‐Liss, Inc.

Effect of noradrenergic lesions on subtypes of alpha 2-adrenoceptors in rat brain.

The binding of [3H]rauwolscine to alpha 2A- (also referred to as alpha 2D-) and alpha 2C-adrenoceptors in homogenates of rat cerebral cortex was measured by exploiting the selectivity of oxymetazoline for alpha 2A-adrenoceptors. Inhibition of [3H]rauwolscine binding by oxymetazoline was modeled best assuming binding to two sites (p < 0.001). Competition curves for oxymetazoline were shifted rightward by the addition of GTP (250 microM) but were still fit best by a two-site model (p < 0.001). A concentration of oxymetazoline was calculated that would optimally antagonize [3H]rauwolscine binding (with GTP present) to oxymetazoline-sensitive alpha 2A-adrenoceptors, minimally inhibiting binding to alpha 2C-adrenoceptors. Subsequently, [3H]rauwolscine binding to alpha 2A- and alpha 2C-adrenoceptors in cortex was examined 3 weeks after destruction of noradrenergic terminals. Binding to alpha 2C-adrenoceptors was increased significantly after treatment with 6-hydroxydopamine, (6-OHDA) compared with vehicle-treated controls, whereas binding to alpha 2A-adrenoceptors was unchanged. Pretreatment of rats with desipramine before 6-hydroxydopamine, to protect noradrenergic neurons, resulted in no changes in binding to either alpha 2A- or alpha 2C-adrenoceptors. Thus, alpha 2C-adrenoceptors are regulated by changes in synaptic availability of norepinephrine. alpha 2A-Adrenoceptors are either not regulated by synaptic norepinephrine or are located both post- and presynaptically so that up-regulation of postsynaptic alpha 2A-adrenoceptors is offset by a loss of presynaptic alpha 2A-adrenoceptors.

Neurochemical Studies of Human Narcolepsy: Alpha-Adrenergic Receptor Autoradiography of Human Narcoleptic Brain and Brainstem

Studies of human and canine narcolepsy-cataplexy syndrome suggest that noradrenergic function may be abnormal. We used quantitative autoradiography to assess noradrenergic alpha-1 and alpha-2 receptors in several regions of seven human narcoleptic and 18 control brains using [3H]prazosin to evaluate alpha-1 receptors, and [3H]UK14304 and [3H]rauwolscine to evaluate alpha-2 receptors. Specific blocking agents were used in combination with the tritiated ligands to assess alpha-1 and alpha-2 receptor subtypes. Although we found few statistically significant differences between narcoleptic and control brains, there were a number of trends. [3H]Prazosin binding to sites in the amygdala, globus pallidus and putamen was reduced by 22-68%, whereas binding was increased by 40% to the inferior olive and by 84% to portions of the dorsal pons. Binding was similar to control values in other regions. In all seven brainstem regions that were evaluated, the ratio of alpha-1b receptor binding to alpha-1a receptor binding was increased. Binding of [3H]UK14304 was increased by 35-74% in the caudate nucleus, putamen and portions of the amygdala and pons. [3H]rauwolscine binding data suggested that increase of alpha-2 receptor binding in the dorsal pons were not due to effects at the imidazole receptor. findings suggest that noradrenergic function may be altered in specific regions of the brain and brainstem in human narcolepsy, although the absence of statistical significance indicates that these trends should be considered preliminary. The trend toward a relative increase of alpha-1b receptor binding in narcoleptic brainstem is consistent with data from studies of canine narcolepsy and suggests that altered activity at this receptor may contribute to the pathogenesis of human narcolepsy. Studies of additional brains will be required to confirm these findings.

Characterization of imidazoline binding protein(s) solubilized from human brainstem: Studies with [3H]idazoxan and [3H]clonidine

Imidazoline binding sites from the human brainstem were solubilized with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS). [3H]idazoxan and [3H]clonidine were used as ligands to characterize the solubilized binding sites. In both the soluble and membrane fractions, [3H]idazoxan binding was saturable, stereoselective, sensitive to imidazolines and insensitive to (-)norepinephrine and to amiloride. The affinities of [3H]idazoxan for the soluble and membrane sites were similar (KD = 25 ± 11 nM and 20 ± 3 nM). In both soluble and membrane fractions, the α2-adrenoceptor binding being masked with (-)norepinephrine, [3H]clonidine bound to a low affinity site which was insensitive to (-)norepinephrine and which exhibited the same selectivity for various drugs as the [3H]idazoxan binding site. α2-adrenoceptor binding was present in the membrane and the soluble fractions although it was difficult to detect in the soluble fraction because of inhibition of [3H]rauwolscine binding by the CHAPS detergent.

Similar ligand binding in recombinant human α2C2-adrenoceptors produced in mammalian, insect and yeast cells

Ligand binding properties were investigated in recombinant human α 2C2-adrenoceptors expressed in three different host systems: Shionogi S115 mouse mammary tumour cells, Spodoptera frugiperda Sf9 insect cells and Saccharomyces cerevisae yeast cells. The expected 43 kDa α 2C2 protein was visualized with immunoblotting using a polyclonal α 2C2-receptor antibody. [ 3H]Rauwolscine binding in cell homogenates or membranes ( B max 3–11 pmol/mg protein; K d approximately 5.5 nM) was inhibited by prazosin, oxymetazoline, RX821002, chlorpromazine and (−)-noradrenaline with and without the GTP-analogue Gpp(NH)p with similar K i values in the different host systems. This indicates that α 2C2-adrenoceptors retain their binding characteristics irrespective of the host environment.

Regulation of adrenergic receptors in the rat kidney.

Chronic daily application of (+/-)-isoprenaline induced a selective-down regulation of beta-adrenoceptors in the kidney: the concentration of [3H]dihydroalprenolol binding sites was significantly lowered by isoprenaline treatment while [3H]prazosin and [3H]rauwolscine binding, representing alpha 1- and alpha 2-adrenoceptors, respectively, was not markedly altered. Since the proportion of high- and low-affinity sites for the non-selective alpha- but relatively beta 1-selective agonist (-)-noradrenaline remained constant and since in [3H]dihydroalprenolol competition experiments the high- and low-affinity site ratio fitted well to the beta 1/beta 2 relation, determined independently by employing ICI 118551 as a beta 2-selective ligand, a parallel decrease of both beta 1- and beta 2-adrenoceptor density can be concluded.

α 2-Adrenoceptors in the guinea-pig uterus: heterogeneity in the circular and longitudinal smooth muscle layers

Homogenate binding and functional studies have been undertaken to investigate the role of α 2-adrenoceptors on the circular and longitudinal myometrial layers of the dioestrous guinea-pig uterus. Each myometrial layer contained a single population of [ 3H]rauwolscine binding sites ( K Dvalues ≈ 3 nM) for which yohimbine exhibited a higher affinity than prazosin, and xylazine a higher affinity than phenylephrine, indicating the presence of α 2-adrenoceptor binding sites. In circular myometrium, xylazine enhanced contractile responses, was more potent than either noradrenaline or phenylephrine in inhibiting forskolin-stimulated accumulation of cyclic AMP, and reduced the inhibitory effect of forskolin on phenylephrine-induced phosphatidyl inositol hydrolysis. In longitudinal myometrium xylazine enhanced contractile responses to phenylephrine but did not inhibit forskolin-stimulated accumulation of cyclic AMP. We conclude that α 2-adrenoceptor binding sites are present in both uterine layers and mediate uterine contractility possibly through different mechanisms.

Age-related changes of noradrenergic-NPY interaction in rat brain: norepinephrine, NPY levels and α-adrenoceptors

Noradrenergic-neuropeptide Y interaction, which is implicated in different physiological functions, was studied in senescent rats. Norepinephrine (NE) and neuropeptide Y (NPY) levels were measured in brainstem and hypothalamus, and α-adrenergic binding was investigated in brainstem in young (4 months) and old (34 months) Wistar rats. NE concentration was the same in senescent rats, whether NPY concentration was decreased both in brainstem and hypothalamus compared to levels in young rats. [ 3H]prazosin binding to α 1-adrenoceptors was not modified, but [ 3H]rauwolscine binding to α 2-adrenoceptors was altered with age. In fact, the density of α 2-adrenoceptors( B max) was lower, while the binding affinity( K d) was increased in old compared to young rats. These results suggest that the decrease of NPY levels could be one of the possible reasons for changes in [ 3H]rauwolscine binding to α 2-adrenoceptors in old rats. The G-protein-adenylate cyclase system, which is impaired in senescent rats, could be involved in the disorganization of noradrenergic-NPY interaction.

Cloning and expression of a fish alpha 2-adrenoceptor.

1. Pigment granule aggregation in specialized cells (melanophores) from the skin of teleost fishes has been shown to be mediated by receptors with an alpha 2-adrenoceptor pharmacology. We now report the cloning of the alpha 2-F, a fish skin alpha 2-receptor from the cuckoo wrasse (Labrus ossifagus). 2. Degenerate oligonucleotides corresponding to conserved regions of the human alpha 2-adrenoceptor subtypes were used in a polymerase chain reaction (PCR) with cDNA prepared from mRNA isolated subtypes were used in a polymerase chain reaction (PCR) with cDNA prepared from mRNA isolated from the skin of the cuckoo wrasse. An 876 base pair (bp) product was obtained that was homologous with that of the human alpha 2-adrenoceptor and was used to screen a genomic library from the cuckoo wrasse. 3. A clone (pTB17BS) consisting of approximately 5 kb of genomic DNA was obtained which contained the nucleotide sequence of the initial PCR product. In addition, it contained an open reading frame that encoded a protein of 432 amino acids and approximately 2 kb of 5'untranslated sequence. The deduced amino acid sequence of this protein showed 47-57% identity with the human alpha 2-adrenoceptors and thus appeared to encode a fish alpha 2-adrenoceptor. 4. In the 5'-untranslated region of the gene, nucleotide sequences were present suggesting that transcription of the alpha 2-F might be regulated by cyclic AMP, calcium and/or steroids. 5. The alpha 2-F was expressed in COS-7 cells and radioligand binding studies were performed with [3H]-rauwolscine. The binding was of high affinity and it was saturable with a KD of 0.8 +/- 0.1 nM and a Bmax of 5.7 +/- 1.0 pmol mg-1 of protein.6. Competition curves for the displacement of specific [3H]-rauwolscine binding showed the following order of potency: for agonists, medetomidine > clonidine >p-aminoclonidine> B-HT 920> (- )-noradrenaline;for antagonists, rauwolscine > atipamezole > yohimbine > phentolamine > prazosin.7. These results show that alpha2-F has characteristics of both the human alpha2-CIO and alpha2-C4 and that it might represent an ancestral alpha2-adrenoceptor subtype.

Postnatal development of central nervous α2-adrenergic binding sites: an in vitro autoradiography study in the tree shrew

The postnatal development of the α 2-adrenoceptor pattern was investigated by in vitro receptor autoradiography with the antagonist [ 3H]rauwolscine in the brains of tree shrews ( Tupaia belangeri). At birth, high numbers of [ 3H]rauwolscine binding sites are diffusely distributed in the whole brain with exception of the neocortex which is very weakly labeled at this time. While the number of [ 3H]rauwolscine binding sites in the cerebellum decreases to low levels during the first three postnatal weeks, several brain regions show a significant increase in binding sites which are progressively concentrated in distinct nuclei. In the medulla oblongata, the diffuse labeling pattern changes so that binding sites become centralized in the dorsomedial nuclei. In the pons, similar changes can be observed with a moderate labeling of the locus coeruleus on postnatal day 10 and a strong labeling in the adult. In the thalamus, a transient appearance of high numbers of [ 3H]rauwolscine binding sites can be observed during the second and third postnatal week in specific nuclei. In the preoptic area and hypothalamus, there are only minor postnatal changes but the numbers of [ 3H]rauwolscine binding sites decrease between postnatal day 5 and adulthood. The high number of binding sites in the limbic system does not significantly change after birth. In the neocortex and the superior colliculus, the [ 3H]rauwolscine labeling pattern which is characteristic for the adult is achieved not before the third postnatal week. Competition experiments demonstrate that [ 3H]RAUW binds with high affinity to α 2-adrenoceptors in the postnatal as well as in the adult brain. Therefore, this study demonstrates region specific developmental profiles of the pattern of α 2-adrenoceptors in the postnatal tree shrew brain.

Effect of incomplete ischemia and reperfusion of the rat brain on the density and affinity of α-adrenergic binding sites in the cerebral cortex. prevention of changes by stobadine and vitamin E.

The effect of incomplete 4 hr ischemia and subsequent 1 hr reperfusion of the rat brain on the density and affinity of α-adrenergic binding sites was investigated. To assess the involvement of oxygen-derived free radicals in the development of ischemic injury, we tested the effect of stobadine and vitamin E on putative changes of the binding parameters of α-adrenergic binding sites in ischemic and reperfused rat brains. Compared to the group of sham operated animals decreased density and increased affinity of [ 3H]dihydroergocryptine binding sites was found in cerebrocortical membranes of rats subjected to 4 hr incomplete ischemia and 1 hr reperfusion. The reduction of B max and K d induced by ischemia and reperfusion of the brain was prevented by stobadine and vitamin E administration. Neither incomplete ischemia nor incomplete ischemia and subsequent reperfusion of the rat brain exerted significant effect on [ 3H]rauwolscine binding to α 2-adrenergic binding sites. Our results suggest that brain ischemia and reperfusion may affect the density and affinity of α 1- rather than of α 2-adrenergic binding sites. The beneficial effect of stobadine and vitamin E indicates that increased generation of oxygen free radicals might play a role in the development of these changes.

Functional analysis of the human α2C-C4 adrenergic receptor in insect cells expressed by a luciferase-based baculovirus vector

A click beetle luciferase-based baculovirus expression vector is described for functional analysis and high level expression of a human α2-adrenergic receptor (α2AR) in Sf9 insect cells. The resultant recombinant baculovirus construct, AcLucGR-α2(C4), was isolated by utilizing the light emitting properties of luciferase and used for abundant expression of the α2C-C4 receptor protein in this lepidopteran insect cell line. A maximal expression of α2-receptors at a level of 1.370 pmol/mg protein was obtained at 48 h after infection as determined by ligand-binding experiments using the α2-receptor antagonist, [ 3H]rauwolscine. The receptor agonists, noradrenaline and clonidine, displaced the [ 3H]rauwolscine binding with K i values 12.3 ± 1.54 μM and 1.23 ± 0.11 μM, respectively. The recombinant receptors were functionally intact since the agonists inhibited forskolin-stimulated cAMP production. Here, however, the maximal inhibition was obtained at 36 h after the infection. The results presented here, suggest that the baculovirus expression vector system (BEVS) provides a simple method for abundant expression of functional α2-receptor subtypes. In addition, co-expression of luciferase proved to be useful for screening and isolation of the recombinant baculovirus.