Comparison of α1-Adrenergic Receptor-Stimulated Inositol Phosphate Formation in Primary Neuronal and Glial Cultures

alpha 1-Adrenergic receptor binding sites and norepinephrine-stimulated 3H-inositol phosphate (3H-InsP) accumulation were measured in primary cultures of neurons and glia from 1-day-old rat brains. The density of alpha 1-adrenergic receptor binding sites was approximately three times higher in membranes from neurons compared to glia. Although norepinephrine was slightly more potent in stimulating 3H-InsP formation in neurons than in glia, the maximal response was greater in glial cells. Norepinephrine-stimulated 3H-InsP formation remained constant for [3H]inositol prelabelling periods of 1-14 days in neurons, whereas the response increased with time in glia and was maximal after 7-10 days of prelabelling. Both the incorporation of [3H]inositol into lipid and basal levels of 3H-InsPs were lower in glial cells than in neurons, which accounted for the greater percent stimulation in glia. Pretreatment with phenoxybenzamine decreased norepinephrine-stimulated 3H-InsP formation in a dose-dependent manner in both neurons and glia by decreasing the maximal response without altering potency. HPLC separation showed that similar types of 3H-InsPs were accumulated in neurons and glial cells. These results demonstrate that alpha 1-adrenergic receptors exist on both neurons and glial cells and activate 3H-InsP accumulation in both cell types. Although receptor density is higher in neurons than in glia, the 3H-InsP response is higher in glia. This difference does not appear to be due to different receptor reserves, but may be due to differential coupling mechanisms in the two cell types.     

α1-Adrenergic Receptors in Neuronal Cultures from Rat Brain: Increased Expression in the Spontaneously Hypertensive Rat

Neuronal cells in primary culture from 1-day-old brains of normotensive, Wistar-Kyoto strain (WKY) and spontaneously hypertensive (SH) rats have been utilized to study the expression of alpha 1-adrenergic receptors. Binding of a selective alpha 1 antagonist, [125I]2-[beta-(4-hydroxy-3-iodophenyl)-ethylaminomethyl]-tetralone ([125I]HEAT) to neuronal membranes prepared from primary brain cultures of WKY and SH rats was 75-80% specific, rapid, and time-dependent although the binding was 1.5-2 times higher in neuronal membranes from SH rat brain cultures. Kinetic analysis of the association and dissociation data demonstrated no significant differences between rat strains. Competition-inhibition experiments provided IC50 values for various antagonists and agonists in the following order: prazosin less than phentolamine less than yohimbine less than phenylephrine less than norepinephrine less than propranolol, suggesting that [125I]HEAT bound selectively to alpha 1-adrenergic receptors. Scatchard analysis of the binding data provided straight lines for both strains of rats, indicating the presence of a homogeneous population of binding sites. It also showed that the increase in the binding in neuronal cells from SH rat brains over those from normotensive WKY controls was a result of an increase in the number of alpha 1-adrenergic receptors. Incubation of neuronal cultures from both strains of rats with phenylephrine, an alpha 1-adrenergic agonist, caused a time- and dose-dependent decrease in the binding of [125I]HEAT. This decrease was due to a decrease in the number of alpha 1-adrenergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

α2-Adrenergic Receptors in Neuronal and Glial Cultures: Characterization and Comparison

Membranes prepared from either neuronal or glial cultures contain alpha 2-adrenergic receptors as determined by the characteristics of [3H]yohimbine [( 3H]YOH) binding. The binding was rapid, reversible, saturable, dependent on the protein concentration used, and reached equilibrium by 5 min in membranes from both neuronal and glial cultures. Scatchard analyses of saturation isotherms revealed similar KD values of 13.7 +/- 1.35 nM (n = 10) for neuronal cultures and 18.42 +/- 2.34 nM (n = 10) for glial cultures. Glial cultures contained many more binding sites for [3H]YOH than neuronal cultures, having a Bmax of 1.6 +/- 0.33 pmol/mg protein (n = 10) compared with 0.143 +/- 0.018 pmol/mg protein (n = 10) in neurons. Drugs selective for alpha 2-adrenergic receptors were the most effective displacers of [3H]YOH binding in both neuronal and glial cultures, i.e., the alpha 2-adrenergic antagonists rauwolscine and yohimbine were better displacers than the other catecholamine antagonists prazosin, corynanthine, or propranolol. The agonists showed the same pattern with the alpha 2-selective drugs clonidine and naphazoline being the most effective competitors for the [3H]YOH site. GTP and its nonhydrolyzable analog. 5'-guanylyl-imidodiphosphate, were able to lower the affinity of the alpha 2-receptors for agonists but not antagonists in membranes from both neuronal and glial cultures, suggesting that the receptors are linked to a G protein in both cell types. The presence of alpha 2-adrenergic receptors in neuronal cultures was also substantiated by light microscopic autoradiography of [3H]YOH binding. In summary, we have demonstrated that both neuronal and glial cultures contain alpha 2-adrenoceptors.     

Antinociceptive Actions of α2-Adrenoceptor Agonists in the Rat Spinal Cord: Evidence for Antinociceptive α2-Adrenoceptor Subtypes and Dissociation of Antinociceptive α2-Adrenoceptors from Cyclic AMP

The antinociceptive actions of intrathecal injections of two alpha 2-adrenergic agonists, UK-14,304 and guanfacine, were investigated in rats after pretreatment of the animals with the noradrenaline neurotoxin N-2-chloroethyl-N-ethyl-2-bromobenzylamine (DSP4) 14 days in advance. The chronic noradrenaline depletion induced by DSP4 caused a marked increase in sensitivity of the antinociceptive action of UK-14,304 in the tail-flick test. By contrast, the antinociceptive effect of guanfacine was not appreciably affected by the DSP4 treatment. The antinociceptive effects of both UK-14,304 and guanfacine were blocked by intraperitoneal injections of yohimbine, a result indicating that both drugs induced their actions by activating alpha 2-adrenoceptors. Both UK-14,304 and guanfacine were found to reduce the production of cyclic AMP (cAMP) in the spinal cord, as determined using an in vitro radioisotopic method. The cAMP inhibitory effects of both agonists were effectively blocked by yohimbine, but not by prazosin, a finding indicating the alpha 2-adrenergic nature of the response. However, the cAMP inhibitory effect of UK-14,304 was not potentiated by pretreatment with DSP4, a finding in marked contrast with the strong potentiation of the antinociceptive action of UK-14,304 induced by the chronic depletion of endogenous noradrenaline. Moreover, intrathecal injections of forskolin, which increased the endogenous levels of spinal cord cAMP fivefold, did not modify the antinociceptive effects of UK-14,304 or guanfacine in neither normal nor DSP4-treated animals. It is suggested that there exist pharmacologically differing alpha 2-adrenergic receptor pathways capable of mediating antinociceptive effects at the level of the spinal cord. The cAMP inhibitory actions of spinal cord alpha 2-adrenoceptors appear not to be directly linked with the antinociceptive actions of these receptors.     

α2-Adrenergic Regulation of Arylalkylamine N-Acetyltransferase in Organ-Cultured Chick Pineal Gland: Characterization with Agonists and Modulation of Experimentally Stimulated Enzyme Activity

In the chicken pineal gland, norepinephrine, released at sympathetic nerve endings, plays a role in synchronizing the circadian rhythm of melatonin synthesis. This effect appears to be exerted via an adrenergic inhibition of arylalkylamine N-acetyltransferase, the melatonin rhythm-generating enzyme. The present study indicates that the nighttime peak of N-acetyltransferase activity developed by organ-cultured chick pineal glands is inhibited by adrenergic agonists with a potency order characterizing alpha 2-adrenergic receptors: UK 14,304 greater than clonidine greater than alpha-methylnorepinephrine = epinephrine greater than cirazoline greater than phenylephrine greater than isoproterenol. The mechanism of this alpha 2-adrenergic response was further analyzed in organ cultures, by studying the ability of clonidine to block the cyclic AMP-dependent and the depolarization-dependent stimulations of N-acetyltransferase activity. Clonidine prevented the rise in N-acetyltransferase activity evoked by the adenylate cyclase activators forskolin and cholera toxin or by the phosphodiesterase inhibitor Ro 20,1724. The stimulatory effect of dibutyryl cyclic AMP was also blocked by clonidine. Activation of pineal alpha 2-adrenergic receptors effectively prevented the stimulation of N-acetyltransferase by depolarizing concentrations of KCl. The possibility that the alpha 2-adrenergic effect might be exerted at a step distal to cyclic AMP production is discussed.     

Regulation of Noradrenaline Release from Rat Occipital Cortex Tissue Chops by α2-Adrenergic Agonists

Noradrenaline (NA) and the alpha 2-adrenergic agonists clonidine, BHT-920, and UK 14304-18 inhibit potassium-evoked release of [3H]NA from rat occipital cortex tissue chops with similar potencies. NA (10(-5) M) was most effective as up to 85% inhibition could be observed compared with 75%, 55%, and 35% for UK 14304-18, clonidine, and BHT-920, respectively, all at 10(-5) M. Potassium-evoked release was enhanced by both forskolin (10(-5) M) and 1 mM dibutyryl cyclic AMP. Pretreatment of tissue chops with 1 mM dibutyryl cyclic AMP in the presence of 3-isobutyl-1-methylxanthine partially reversed the alpha 2-adrenergic agonist inhibition of NA release. No reversal of inhibition was observed following pretreatment with 10(-5) M forskolin. The effects of clonidine, BHT-920, UK-14308-18, and NA on cyclic AMP formation stimulated by (a) forskolin, (b) isoprenaline, (c) adenosine, (d) potassium, and (e) NA were examined. Only cAMP formation stimulated by NA was inhibited by these alpha 2-adrenergic agonists. These results suggest that only a small fraction of adenylate cyclase in rat occipital cortex is coupled to alpha 2-adrenergic receptors. These results are discussed in relation to recent findings that several alpha 2-adrenergic receptor subtypes occur, not all of which are coupled to the inhibition of adenylate cyclase, and that alpha 2-adrenergic receptors inhibit NA release in rat occipital cortex by a mechanism that does not involve decreasing cyclic AMP levels.     

Noradrenergic Inhibition and α2-Adrenergic Stimulation of Melatonin Secretion in the Pigeon

Adrenergic regulatory mechanisms of melatonin synthesis and secretion were studied in the pigeon in vivo. Late-afternoon intraperitoneal injection of noradrenaline (NA; 1 mg/kg) resulted in a significant decrease in plasma melatonin levels in 3 h. The same effect was seen after phenylephrine treatment (1 mg/kg i.p.), indicating that an alpha 1-adrenergic mechanism may mediate the inhibition. Propranolol treatment had no effect on plasma melatonin levels, supporting this concept. Detomidine (1 mg/kg i.p.), an alpha 2-adrenergic agonist, increased melatonin levels. This stimulatory effect was blocked by yohimbine, an alpha 2-adrenergic antagonist. However, yohimbine alone had no effect on the plasma melatonin levels, suggesting that alpha 2-adrenergic transmission is not primarily responsible for the nocturnal stimulation of melatonin synthesis and secretion in the pigeon.     

α1-Adrenergic Receptor-Mediated Downregulation of Angiotensin II Receptors in Neuronal Cultures

Previous evidence has suggested that brain catecholamine levels are important in the regulation of central angiotensin II receptors. In the present study, the effects of norepinephrine and 3,4-dihydroxyphenylethylamine (dopamine) on angiotensin II receptor regulation in neuronal cultures from rat hypothalamus and brainstem have been examined. Both catecholamines elicit significant decreases in [125I]angiotensin II-specific binding to neuronal cultures prepared from normotensive rats, effects that are dose dependent and that are maximal within 4-8 h of preincubation. Saturation and Scatchard analyses revealed that the norepinephrine-induced decrease in the binding is due to a decrease in the number of angiotensin II receptors in neuronal cultures, with little effect on the receptor affinity. Norepinephrine has no significant actions on [125I]angiotensin II binding in cultures prepared from spontaneously hypertensive rats. The downregulation of angiotensin II receptors by norepinephrine or dopamine is blocked by alpha 1-adrenergic and not by other adrenergic antagonists, a result suggesting that this effect is initiated at the cell surface involving alpha 1-adrenergic receptors. This is further supported by our data indicating a parallel downregulation of specific alpha 1-adrenergic receptors elicited by norepinephrine. In summary, these results show that norepinephrine and dopamine are able to alter the regulation of neuronal angiotensin II receptors by acting at alpha 1-adrenergic receptors, which is a novel finding.     

N-Acetylation of Serotonin Is Correlated with α2- but Not with β-Adrenergic Regulation of Cyclic AMP Levels in Cultured Chick Pineal Cells

We investigated the role of cyclic AMP (cAMP) in alpha 2- and possible beta-adrenergic regulation of arylalkylamine-N-acetyltransferase (NAT), the penultimate enzyme in the biosynthesis of melatonin. The study was performed on primary cultures of dispersed chick pineal cells. Electron microscopy indicated that approximately 70% of the dispersed cells were modified photoreceptors. A similar proportion of melatoninergic cells was detected by immunocytochemical labeling of hydroxyindole-O-methyltransferase, the final enzyme in the biosynthesis of melatonin. Adrenergic agonists caused a sustained 50% inhibition of forskolin-augmented cAMP levels and NAT activity, with an alpha 2-adrenergic potency order of UK 14,304 greater than or equal to clonidine greater than norepinephrine greater than phenylephrine. Noradrenergic inhibition of 3-isobutyl-1-methylxanthine-augmented cAMP levels and NAT activity was reversed by yohimbine (an alpha 2-adrenergic antagonist) but not by prazosin (an alpha 1-adrenergic antagonist). The alpha-adrenergic inhibition of cAMP accumulation and NAT activity was prevented by pertussis toxin. Addition of propranolol (a beta-adrenergic antagonist) was necessary to observe an inhibitory effect of norepinephrine on cAMP levels but not on NAT activity. Similarly, the beta-adrenergic agonist isoproterenol transiently increased cAMP levels but did not affect NAT activity. The data indicate that the alpha 2-adrenergic inhibition of NAT activity in chick pineal cells is strongly correlated with an inhibition of cAMP accumulation. The lack of beta-adrenergic effect on NAT suggests that beta-adrenoceptors might be on a subset of cells that do not produce melatonin or that the beta-adrenergic-induced increase in cAMP levels is too transient to affect NAT.     

α2-Adrenergic and Muscarinic Cholinergic Receptors Have Oposing Actions on Cyclic AMP Levels in SK-N-SH Human Neuroblastoma Cells

Forskolin and vasoactive intestinal polypeptide (VIP) were shown to increase cyclic AMP accumulation in a human neuroblastoma cell line, SK-N-SH cells. The alpha 2-adrenergic agonist UK 14304 decreased forskolin-stimulated cyclic AMP levels by 40 +/- 2%, with an EC50 of 83 +/- 20 nM. This response was blocked by pretreatment with pertussis toxin (PT) (EC50 = 1 ng/ml) or by the alpha 2-antagonists yohimbine, idazoxan, and phentolamine. Antagonist IC50 values were 0.3 +/- 0.1, 2.2 +/- 0.3, and 1.4 +/- 0.1 microM, respectively. This finding suggests the presence of normal inhibitory coupling of SK-N-SH cell alpha 2-adrenergic receptors to adenylate cyclase via the inhibitory GTP-binding protein species, Gi. Muscarinic receptors in many target cell types are coupled to inhibition of adenylate cyclase. However, in SK-N-SH cells, muscarinic agonists synergistically increased (67-95%) the level of cyclic AMP accumulation elicited by forskolin or VIP. EC50 values for carbamylcholine (CCh) and oxotremorine facilitation of the forskolin response were 1.2 +/- 0.2 and 0.3 +/- 0.1 microM, respectively. Pharmacological studies using the muscarinic receptor subtype-preferring antagonists 4-diphenylacetoxy-N-methylpiperidine, pirenzepine, and AF-DX 116 indicated mediation of this response by the M3 subtype. IC50 values were 14 +/- 1, 16,857 +/- 757, and 148,043 +/- 16,209 nM, respectively. CCh-elicited responses were unaffected by PT pretreatment. Muscarinic agonist binding affinity was indirectly measured by the ability of CCh to compete for [3H]quinuclidinyl benzilate binding sites on SK-N-SH cell membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of Muscarinic and of α1-Adrenergic Receptors on Astrocytes Results in the Accumulation of Inositol Phosphates

Astrocyte-enriched cultures prepared from the newborn rat cortex incorporated [3H]myo-inositol into intracellular free inositol and inositol lipid pools. Noradrenaline and carbachol stimulated the turnover of these pools resulting in an increased accumulation of intracellular [3H]inositol phosphates. The effects of noradrenaline and carbachol were dose-dependent and blocked by specific alpha 1-adrenergic and muscarinic cholinergic receptor antagonists, respectively. The increase in [3H]inositol phosphate accumulation caused by these receptor antagonists was virtually unchanged when cultures were incubated in Ca2+-free medium, but was abolished when EGTA was also present in the Ca2+-free medium. Cultures of meningeal fibroblasts, the major cell type contaminating the astrocyte cultures, also accumulated [3H]myo-inositol, but no increased accumulation of [3H]inositol phosphates was found in response to either noradrenaline or carbachol.     

See-Saw Signal Processing in Pinealocytes Involves Reciprocal Changes in the α1-Adrenergic Component of the Cyclic GMP Response and the β-Adrenergic Component of the Cyclic AMP Response

Pineal cyclic AMP and cyclic GMP are regulated by norepinephrine (NE) acting through alpha 1- and beta-adrenoceptors. beta-Adrenergic stimulation appears to be an absolute requirement and alpha 1-adrenergic activation amplifies beta-adrenergic stimulation of the cyclic AMP response 10-fold and the cyclic GMP response 100-fold, respectively. Chronic deprivation of adrenergic stimulation, due to exposure to constant light (LL) or by surgical denervation, enhances the cyclic AMP response and diminishes the cyclic GMP response as compared to control animals in a 10:14 light/dark (LD) cycle. This phenomenon is termed see-saw signal processing. In the current study we find these changes do not reflect shifts in the time course or Ka of these responses. Dose-response studies indicate the beta-adrenergic component of cyclic AMP stimulation is enhanced and the alpha 1-adrenergic component of cyclic GMP stimulation is diminished in LL pinealocytes. Several observations indicate these changes may reflect alterations in Ca2+-sensitive postreceptor mechanisms.     

Identification of α2-Adrenergic Receptors in Chicken Pineal Gland Using [3H]Rauwolscine

The norepinephrine-induced inhibition of avian pineal N-acetyltransferase activity appears to be mediated by alpha 2-adrenergic receptors. In this study, alpha 2-adrenergic receptors in the chicken pineal gland were directly identified by radioligand binding. Membrane preparations of pineal glands from chickens from 1 to 6 weeks of age were examined using [3H]rauwolscine, a selective alpha 2-adrenergic receptor antagonist, to characterize the binding sites. The results indicate no ontological change in either the affinity (KD) or density of receptor binding sites (Bmax) during the time span examined. The binding was saturable and of high affinity with a mean KD of 0.27 +/- 0.01 nM and a mean Bmax of 242 +/- 12 fmol/mg protein. Further characterization of these binding sites indicated that the alpha 2-adrenergic receptor is of the alpha 2A subtype, since prazosin and ARC-239 bound with low affinities and oxymetazoline bound with high affinity.     

Solubilization and Characterization of Rat Brain α2-Adrenergic Receptor

alpha 2-Adrenergic receptors labelled by [3H]-clonidine (alpha 2-agonist) can be solubilized from the rat brain in a form sensitive to guanine nucleotides with a zwitterionic detergent, 3-[3-(cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS). About 40% of the original [3H]CLO binding sites in the membranes were solubilized with 6 mM CHAPS. Separation of the soluble [3H]CLO-bound complex was performed by the vacuum filtration method using polyethylenimine-treated GF/B filters. Solubilized [3H]CLO binding sites retained the same pharmacological characteristics of membrane-bound alpha 2-adrenergic receptors. Scatchard plots of [3H]CLO binding to solubilized alpha 2-receptors were curvilinear, indicating the existence of the two distinct binding components. Solubilized receptors were eluted as a single peak from Bio-Gel A-1.5 m column with a Stokes radius of 6.6 nm. The isoelectric point was 5.6-5.8. Regulations of the receptor binding by guanine nucleotides, monovalent cations, and sulfhydryl-reactive agents were maintained intact in the soluble state, whereas those by divalent cations were lost. The apparent retention of receptors and guanine nucleotide binding regulatory component(s) in the soluble state may allow a investigation of the regulation mechanisms of the brain alpha 2-adrenergic receptor system at the molecular level.     

Characterization and Regulation of β1-Adrenergic Receptors in a Human Neuroepithelioma Cell Line

Intact human neuroepithelioma SK-N-MC cells bound the beta-adrenergic antagonist (-)-[3H]-CGP 12177 with a KD of 0.13 nM and a Bmax of 17,500 sites/cell. When the cells were exposed to beta-adrenergic agonists, they accumulated cyclic AMP in the following order of potency: isoproterenol much greater than norepinephrine greater than epinephrine, which is indicative of a beta 1-subtype receptor. Membranes prepared from the cells bound (-)-3-[125I]iodocyanopindolol with a KD of 11.5 pM. Inhibition of agonist-stimulated cyclic AMP production and competition binding experiments indicated that the beta 1-selective antagonists CGP 20712A and ICI 89,406 were much more potent than the beta 2-selective antagonist ICI 118,551. Analysis of the displacement curves indicated that the cells contained only beta 1-adrenergic receptors. Northern blot analysis of SK-N-MC mRNA using cDNA probes for the beta 1- and beta 2-adrenergic receptors revealed the presence of a very strong beta 1-adrenergic receptor mRNA signal, while under the same conditions no beta 2-adrenergic receptor mRNA was observed. Thus, SK-N-MC cells appear to express a pure population of beta 1-adrenergic receptors. When the cells were exposed to isoproterenol, there was no observable desensitization during the first hour. After longer exposure, desensitization slowly occurred and the receptors slowly down-regulated to 50% of control levels by 24 h. Other agents that elevate cyclic AMP levels, such as forskolin, cholera toxin, and cyclic AMP analogues, caused no or little substantial receptor loss.     

Vasoactive Intestinal Peptide Stimulates Chick Pineal Melatonin Production and Interacts with Other Stimulatory and Inhibitory Agents but Does Not Show α1-Adrenergic Potentiation

Vasoactive intestinal peptide (VIP) is known to mimic the effects of beta-adrenergic receptor stimulation in the rat pineal, including marked potentiation by alpha 1-adrenergic receptor stimulation, and to cause increased melatonin synthesis. In contrast, the chick pineal does not respond to beta-adrenergic stimulation, and melatonin synthesis is inhibited by norepinephrine via an alpha 2-adrenergic receptor. The present experiments show that chick pineal cells in primary culture do, however, respond to VIP with increased melatonin production. The effect of VIP was inhibited by addition of norepinephrine or of nitrendipine or by exposing the cells to "unexpected" white light. Stimulation by VIP was enhanced by addition of forskolin or Bay K 8644 but not by alpha 1-adrenergic receptor stimulations. Although stimulation by VIP appears similar in the chick pineal to that seen in the rat pineal and other systems, "dual-receptor regulation," at least with alpha 1-adrenergic receptors, appears to be absent.     

Ligand recognition of serine-cysteine amino acid exchanges in transmembrane domain 5 of alpha2-adrenergic receptors by UK 14,304

Ligand binding of UK 14,304 reveals notable species (i.e., human-rodent) and receptor-subtype differences of alpha2-adrenergic receptors (alpha2-ARs). To study the molecular basis of the selectivity of UK 14,304, we compared a series of conservative serine-cysteine exchange mutants at ligand-accessible positions in transmembrane domain 5 of the human and mouse alpha2A-ARs. UK 14,304 bound with approximately 200-fold higher affinity to the human alpha2A-AR wild-type receptor compared with the human alpha2A-ARSer201 mutant, but only an approximately fivefold difference was seen with the corresponding mouse alpha2A-AR variant. These effects of cysteine-serine exchanges only involved the agonist low-affinity forms of the receptors, as the affinity of [3H]UK 14,304 for the agonist high-affinity receptor populations was not influenced. The apparent affinities of a set of eight structurally diverse alpha2-AR ligands (six agonists and two antagonists) were not influenced significantly by the cysteine-serine exchanges (except for oxymetazoline and yohimbine, with up to nine- and eightfold differences in affinity, respectively). We conclude that position 201 (a) plays a primary role in determining observed subtype/species selectivity of UK 14,304 in competitive antagonist radioligand binding assays and (b) does not determine the subtype selectivity of chlorpromazine.     

Coupling of Astroglial α2-Adrenoreceptors to Second Messenger Pathways

Abstract: We have investigated which α₂-receptor subtypes are expressed in cultured cortical astroglia, and their coupling to second messengers. Binding assays using [³H]rauwolscine showed a very low number of α₂ receptors in the astrocytic cultures. Treatment of cultures with dibutyryl cyclic AMP (dBcAMP) increased significantly the number of receptors. The RNase protection assay was used to investigate which receptor subtype the cells express. The α_2B message was expressed at a low level in both treated and untreated cells, the levels of mRNA for the α_2A/D subtype were up-regulated significantly in cells treated with dBcAMP and no expression of mRNA for the α_2C subtype was detected. The α₂ agonist dexmedetomidine inhibited forskolin-induced increases in cyclic AMP both in treated and untreated cultures in a pertussis toxin-dependent manner. This effect was abolished by the α₂-receptor antagonist rauwolscine. Selective α₂-receptor agonists dexmedetomidine, clonidine, and UK14,304 all increased intracellular calcium only in dBcAMP-treated cells. The antagonist rauwolscine abolished this effect. Ca²⁺ responses were also seen in the absence of extracellular Ca²⁺ and they were inhibited by the phospholipase C inhibitor U-73122, suggesting that astroglial α₂ receptors are coupled to the inositol phospholipid pathway. We therefore also tested the effect of dexmedetomidine directly on inositol 1,4,5-trisphosphate accumulation. A significant increase was seen that was blocked by the antagonist rauwolscine and, as expected, by U-73122. In short, the results demonstrate that the α₂ receptors in astroglia are coupled to multiple second messenger pathways. They are up-regulated in cells treated with dBcAMP, which simultaneously assume a process-bearing morphology. If this morphological change reflects some in vivo process such as reactive gliosis, the up-regulation of α₂-receptor expression could mean an adaptive change in astrocytic responses to a common neurotransmitter, noradrenaline.     

Electroconvulsive Shock Increases α1b-but Not α1a-Adrenoceptor Binding Sites in Rat Cerebral Cortex

Repeated administration of electroconvulsive shock (ECS) increases [3H]prazosin binding to alpha 1-adrenoceptors in rat cerebral cortex. In contrast, [3H]WB4101 binding in cortex has been reported to be unchanged after ECS. [3H]Prazosin labels two alpha 1-adrenoceptor subtypes, termed alpha 1a and alpha 1b, whereas [3H]WB4101 labels the alpha 1a subtype preferentially. The purpose of this study was to determine whether ECS increases one or both alpha 1-adrenoceptor subtypes in rat cerebral cortex. We found that treatment of rats with ECS once daily for 10-12 days increased [3H]prazosin binding in cortex by about 25% but did not significantly alter [3H]WB4101 binding to alpha 1-adrenoceptors. Measurement of alpha 1a and alpha 1b receptors by competition analysis of the selective alpha 1a antagonist 5-methylurapidil against [3H]prazosin and measurement of [3H]prazosin binding in homogenates preincubated with chlorethylclonidine, which alkylates alpha 1b binding sites, also indicated that the ECS-induced increase in alpha 1-adrenoceptors is confined to the alpha 1b subtype. In contrast to its effect on [3H]prazosin binding, ECS did not increase phosphoinositide hydrolysis as measured by [3H]inositol 1-phosphate accumulation in slices of rat cerebral cortex stimulated by either norepinephrine or phenylephrine. The failure of ECS to increase [3H]inositol 1-phosphate accumulation stimulated by phenylephrine, which is a partial agonist for this response, suggests that spare receptors do not account for the apparent absence of effect of ECS on alpha 1-adrenoceptor-mediated phosphoinositide hydrolysis.     

Multiple Adrenergic Receptor Subtypes Controlling Cyclic AMP Formation: Comparison of Brain Slices and Primary Neuronal and Glial Cultures

The adrenergic receptor subtypes involved in cyclic AMP responses to norepinephrine (NE) were compared between slices of rat cerebral cortex and primary neuronal and glial cultures from rat brain. In neuronal cultures, NE and the beta-adrenergic receptor agonist isoproterenol (ISO) caused similar increases in cyclic AMP, which were not altered by the alpha-adrenergic receptor antagonist phentolamine. In glial cultures, NE caused a much smaller cyclic AMP response than did ISO, and this difference was reversed by alpha-adrenergic receptor antagonists (phentolamine greater than yohimbine greater than prazosin). alpha 2-Adrenergic receptor agonists partially inhibited the ISO response in glial cultures to a level similar to that observed with NE alone (clonidine = UK 14,304 greater than NE greater than 6-fluoro-NE greater than epinephrine). In slices from cerebral cortex, NE caused a much larger increase in cyclic AMP than did ISO, and this difference was reversed by alpha-adrenergic receptor antagonists with a different order of potency (prazosin greater than phentolamine greater than yohimbine). alpha 1-Adrenergic receptor agonists potentiated the response to ISO to a level similar to that observed with NE alone (epinephrine = NE greater than phenylephrine greater than 6-fluoro-NE greater than methoxamine). In all three tissue preparations, large responses to both alpha 1-receptor activation (increases in inositol phosphate accumulation) and alpha 2-receptor activation (decreases in forskolin-stimulated cyclic AMP accumulation) were observed. These data indicate that all of the major adrenergic receptor subtypes (beta, alpha 1, alpha 2) are present in each tissue preparation.(ABSTRACT TRUNCATED AT 250 WORDS)