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.     

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.     

Differential Involvement of the Arachidonic Acid Cascade on the α1-Adrenergic Potentiation of Vasoactive Intestinal Peptide-Versus β-Adrenergic-Stimulated Cyclic AMP and Cyclic GMP Accumulation in Rat Pinealocytes

In the rat pineal gland, alpha 1-adrenergic agonists, which stimulate arachidonic acid release, also potentiate vasoactive intestinal peptide (VIP)- or beta-adrenergic-stimulated cyclic AMP (cAMP) and cyclic GMP (cGMP) accumulation. In this study, the possible involvement of the arachidonic acid pathway in the potentiation mechanism was examined in dispersed rat pinealocytes using two inhibitors of the arachidonic acid cascade, indomethacin and nordihydroguaiaretic acid. These two inhibitors appeared to have differential effects on the alpha 1-adrenergic potentiation of VIP- or beta-adrenergic-stimulated cAMP and cGMP responses. Whereas nordihydroguaiaretic acid was effective in suppressing both the alpha 1-adrenergic potentiation of VIP- or beta-adrenergic-stimulated cAMP and cGMP responses, indomethacin inhibited selectively the VIP-mediated cAMP and cGMP responses. The role of arachidonic acid metabolites was further determined using several prostaglandins--A2, I2, E2, and F2 alpha--and leukotrienes--B4, C4, and D4. Of the seven compounds tested, prostaglandins E2 and F2 alpha stimulated basal cAMP but not cGMP accumulation. The prostaglandin E2- and F2 alpha-stimulated cAMP responses were additive to those stimulated by VIP or beta-adrenergic receptors. The other five compounds had no effects on basal or VIP- or beta-adrenergic-stimulated cAMP or cGMP accumulation. Taken together, these findings indicate that the arachidonic acid cascade is likely involved in the alpha 1-adrenergic potentiation of VIP- or beta-adrenergic-stimulated cAMP and cGMP accumulation. However, the specific arachidonic acid metabolite involved in the potentiation mechanisms of VIP- versus beta-adrenergic-stimulated cyclic nucleotide responses may be different.     

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-Adrenoceptor-Mediated Inhibition of Electrically Evoked [3H]Noradrenaline Release from Chick Sympathetic Neurons: Role of Cyclic AMP

Abstract: This study explores the role of cyclic AMP in electrically evoked [³H]noradrenaline release and in the α₂-adrenergic modulation of this release in chick sympathetic neurons. Along with an increase in stimulation-evoked tritium overflow, applications of forskolin enhanced the formation of intracellular cyclic AMP. Both effects of forskolin were potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. The forskolin-induced increase in overflow was abolished by the Rp-diastereomer of cyclic AMP-thioate, an antagonist at cyclic AMP-dependent protein kinases, and 1,9-dideoxy-forskolin, an inactive analogue at adenylyl cyclase, had no effect on the evoked overflow. A 24-h pretreatment with either cholera toxin or forskolin reduced the subsequent forskolin-induced accumulation of cyclic AMP and inhibited the stimulation-evoked release. Basal cyclic AMP production, however, remained unaltered after forskolin treatment and was enhanced after 24 h of cholera toxin exposure. The α₂-adrenergic agonist bromoxidine did not affect the formation of cyclic AMP stimulated by forskolin but reduced electrically evoked release. However, effects of bromoxidine on ³H overflow were attenuated by forskolin as well as by 8-bromo-cyclic AMP. Effects of bromoxidine on [³H]noradrenaline release were paralleled by an inhibition of voltage-activated Ca²⁺ currents, primarily through a delayed time course of current activation. This effect was abolished when either forskolin or 8-bromo-cyclic AMP was included in the pipette solution. Both substances, however, failed to affect Ca²⁺ currents in the absence of bromoxidine. These results suggest that the signaling cascade of the α₂-adrenergic inhibition of noradrenaline release involves voltage-activated Ca²⁺ channels but not cyclic AMP. Elevated levels of cyclic AMP, however, antagonize this α₂-adrenergic reduction, apparently through a disinhibition of Ca²⁺ channels.     

Cyclic AMP Analogues Potentiate k-Opiate and Attenuate α2-Adrenoceptor Agonist Effects on Intrasynaptosomal Free Calcium

The intrasynaptosomal free calcium concentration ([Ca2+]i) was measured in quin2-loaded synaptosomes prepared from rat cerebral cortex. Membrane-permeant cyclic adenosine-3',5'-monophosphate (cAMP) analogues [8-bromo-cyclic adenosine-3',5'-monophosphate (8-Br-cAMP) and dibutyryl-cyclic adenosine-3',5'-monophosphate (db-cAMP)] increased [Ca2+]i in a dose-dependent manner; The maximal increases were approximately 50% for 8-Br-cAMP and 35% for db-cAMP and occurred at approximately 10 microM with both analogues. Clonidine (1 microM) alone reduced [Ca2+]i by 26.5%; db-cAMP and 8-Br-cAMP attenuated this reduction to 14.2 and 8.2%, respectively. In contrast, the reduction (19.9%) in [Ca2+]i induced by the preferential kappa-opiate agonist dynorphin A(1-13) was not attenuated by the cAMP analogues; in fact, db-cAMP and 8-Br-cAMP potentiated the effect of dynorphin A(1-13) (1 microM), producing decreases in [Ca2+]i of 33.6 and 29.6%, respectively. We conclude that although alpha 2-adrenergic and kappa-opiate receptors both reduce [Ca2+]i, the alpha 2-adrenoceptor-mediated response and the kappa-opiate receptor-mediated response involve different effector mechanisms. It appears that presynaptic alpha 2-adrenoceptor agonist effects are linked to reductions in adenylate cyclase activity and cAMP production and a resultant increase in Ca2+ sequestration, Ca2+-channel blockade, or both. On the other hand, the kappa-opiate-mediated effects possibly involve an increase in cAMP production and a blockade of Ca2+ entry.     

GABAB Receptor-Mediated Enhancement of Vasoactive Intestinal Peptide-Stimulated Cyclic AMP Production in Slices of Rat Cerebral Cortex

Basal and vasoactive intestinal peptide (VIP)-stimulated accumulations of cyclic AMP were measured in slices of rat cerebral cortex. Neither gamma-aminobutyric acid (GABA) nor the selective GABAB receptor agonist (-)-baclofen stimulated basal cyclic AMP accumulation, whereas VIP caused a large dose-dependent increase in cyclic AMP levels. However, in the presence of 100 microM (-)-baclofen, the effects of VIP on cyclic AMP accumulation were significantly enhanced, with the responses to 1 microM and 10 microM VIP being approximately doubled. The enhancing effects of (-)-baclofen was dose related (1-1,000 microM), but an enhancing effect was not observed with 100 microM (+)-baclofen. In the presence of the GABA uptake inhibitor nipecotic acid (1 mM), GABA caused a similar dose-related enhancement of the VIP response. The ability of either GABA or (-)-baclofen to augment VIP-stimulated production of cyclic AMP was not mimicked by the GABAA, agonists isoguvacine and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) and was not antagonized by the GABAA antagonist bicuculline. The putative GABAB antagonist 5-aminovaleric acid (1 mM) significantly reduced the effect of (-)-baclofen. The ability of (-)-baclofen to enhance VIP-stimulated accumulation of cyclic AMP was observed in slices of rat cerebral cortex, hippocampus, and hypothalamus. These results indicate that GABA and (-)-baclofen can enhance VIP-stimulated accumulation of cyclic AMP in rat brain slices via an interaction with specific GABAB receptors.     

Effect of Noradrenergic Lesions on Subtypes of α2-Adrenoceptors in Rat Brain

Abstract: The binding of [³H]rauwolscine to α_2A- (also referred to as α_2D-) and α_2C-adrenoceptors in homogenates of rat cerebral cortex was measured by exploiting the selectivity of oxymetazoline for α_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 µM) 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 α_2A-adrenoceptors, minimally inhibiting binding to α_2C-adrenoceptors. Subsequently, [³H]rauwolscine binding to α_2A- and α_2C-adrenoceptors in cortex was examined 3 weeks after destruction of noradrenergic terminals. Binding to α_2C-adrenoceptors was increased significantly after treatment with 6-hydroxydopamine (6-OHDA) compared with vehicle-treated controls, whereas binding to α_2A-adrenoceptors was unchanged. Pretreatment of rats with desipramine before 6-hydroxydopamine, to protect noradrenergic neurons, resulted in no changes in binding to either α_2A- or α_2C-adrenoceptors. Thus, α_2C-adrenoceptors are regulated by changes in synaptic availability of norepinephrine. α_2A-Adrenoceptors are either not regulated by synaptic norepinephrine or are located both post- and presynaptically so that up-regulation of postsynaptic α_2A-adrenoceptors is offset by a loss of presynaptic α_2A-adrenoceptors.     

Modulation of Histamine Release and Synthesis in the Brain Mediated by α2-Adrenoceptors

Abstract: The adrenergic regulation of histamine release was studied in rat brain slices labeled with L-[³H]histidine. Noradrenaline in increasing concentrations progressively inhibited K⁺-evoked [³H]histamine release from cortical slices, whereas phenylephrine and isoprenaline were ineffective. Yohimbine, a preferential α2-adrenoceptor antagonist, reversed the noradrenaline effect in an apparently competitive manner and with a mean K i value of 30 n M . Phentolamine reversed the noradrenaline effect with a similar potency, whereas propranolol was ineffective. The imidazolines clo-nidine and oxymetazoline acted as partial agonists, oxymeta-zoline even behaving as an apparent antagonist. In vivo clo-nidine also inhibited [³H]histamine formation in cerebral cortex, an effect reversed by the administration of yohimbine. However, yohimbine failed to increase significantly [³H]histamine release in vitro and [³H]histamine formation in vivo, suggesting that adrenergic receptors are not activated by endogenous noradrenaline released under basal conditions. It is concluded that adrenergic α₂-adrenoceptors presumably located on histaminergic axons control release and synthesis of histamine in the brain.     

Astrocytes from Forebrain, Cerebellum, and Spinal Cord Differ in Their Responses to Vasoactive Intestinal Peptide

Astrocytes from cortex, cerebellum, and spinal cord responded to isoproterenol and vasoactive intestinal peptide (VIP) with increases in intracellular cyclic AMP levels. The response to VIP was as great as that to isoproterenol in cortical astrocytes (180-fold and 185-fold, respectively), and the effect of VIP in combination with isoproterenol was partially additive. Spinal cord astrocytes also responded to VIP and isoproterenol with equal potency (seven- to ninefold and eight- to 13-fold, respectively), but the level of response was much smaller than in cortex. Spinal cord astrocytes were synergistic in their response to VIP and isoproterenol. The response to VIP was lowest in cerebellar astrocytes (only threefold), and no additivity was observed when VIP was added together with isoproterenol. A small response to alpha-melanocyte stimulating hormone (alpha-MSH) was also observed in cortex and cerebellum, but not in spinal cord. Somatostatin inhibited the response to isoproterenol in cortex and cerebellum, but had no effect in spinal cord. The results from the above study show that astrocytes obtained from these three regions of the rat CNS express quite different responses to VIP and alpha-MSH and further point to possible astrocyte heterogeneity.     

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)     

No Role for Phospholipase A2 and Protein Kinase C in the Potentiation by α-Adrenoceptors of β-Adrenoceptor-Mediated Cyclic AMP Formation in Rat Brain

This study was undertaken to examine the role of phospholipase A2 and protein kinase C in the potentiation of beta-adrenoceptor-mediated cyclic AMP formation by alpha-adrenoceptors in rat cerebral cortical slices. Inhibition of arachidonic acid metabolism by a range of cyclooxygenase and lipoxygenase inhibitors had no effect on the potentiation of isoprenaline-stimulated cyclic AMP. Conversely, stimulation of leukotriene formation had no effect on the response to isoprenaline. The phospholipase A2 activator, melittin, stimulated cyclic AMP and potentiated the effect of isoprenaline, but these responses were not influenced by cyclooxygenase or lipoxygenase inhibitors. Indomethacin was also ineffective against the potentiation of vasoactive intestinal peptide-stimulated cyclic AMP by noradrenaline. Phorbol ester potentiated the cyclic AMP response to isoprenaline, and this potentiation was antagonized by three different putative protein kinase C inhibitors. However, the same inhibitors did not affect the alpha-adrenoceptor-stimulated enhancement of the response to isoprenaline. We have found no evidence, therefore, to support the suggestion that arachidonic acid and its metabolites and/or protein kinase C mediate the alpha-adrenoceptor modulation of beta-adrenoceptor function.     

Presynaptic α2 Adrenoceptors Inhibit Glutamate Release from Rat Spinal Cord Synaptosomes

Abstract: The presynaptic regulation of amino acid release from nerve terminals was investigated using synaptosomes prepared from the rat spinal cord. The basal releases of endogenous glutamate (Glu), aspartate (Asp), and γ-amino-butyric acid (GABA) were 34.6, 21.5, and 10.0 pmol/min/mg of protein, respectively. Exposure to a depolarizing concentration of KCl (30 m M ) evoked 2.7-, 1.5-, and 2.9-fold increases in Glu, Asp, and GABA release, respectively. Clonidine reduced the K⁺-evoked overflow of Glu to 56% of the control overflow with a potency (IC₅₀) of 17 n M , but it did not affect K⁺-evoked overflow of Asp, GABA, and their basal releases. Similarly, noradrenaline inhibited the K⁺-evoked overflow of Glu, although phenylephrine and isoproterenol showed no effect. The inhibitory effect of clonidine was counteracted by α₂-adrenoceptor antagonists, rauwolscine, yohimbine, and idazoxan, regardless of the imidazoline structures. Because Glu is considered a neurotransmitter of primary afferents that transmit both nociceptive and nonnociceptive stimuli in the spinal cord, these data suggest that part of Glu release may be regulated by the noradrenergic system through α₂ adrenoceptors localized on the primary afferent terminals.     

Effects of Vasoactive Intestinal Peptide and Other Peptides on Cyclic AMP Accumulation in Intact Pieces and Isolated Horizontal Cells of the Teleost Retina

The effects of vasoactive intestinal peptide (VIP) and several other peptides have been examined on cyclic AMP accumulation in intact pieces and isolated horizontal cells of the teleost (carp) retina. VIP was the most effective peptide examined, inducing a dose-related response, and an approximately fivefold increase in cyclic AMP production when used at a concentration of 10 microM. Porcine histidine isoleucine-containing peptide and secretin, peptides structurally related to VIP, also stimulated cyclic AMP accumulation, but at concentrations of 10 microM induced responses which were only approximately 40% and 10%, respectively, of the response observed with 10 microM VIP. In contrast, several other peptides, including glucagon, neurotensin, somatostatin, luteinizing hormone-releasing hormone, alpha-melanocyte-stimulating hormone, cholecystokinin octapeptide26-33, gastrin-releasing peptide, thyrotropin-releasing hormone, and VIP10-28 were totally inactive. The response to 10 microM VIP was not antagonized by several dopamine antagonists, indicating the presence of a population of specific VIP receptors coupled to adenylate cyclase, distinct from the population of dopamine receptors coupled to adenylate cyclase also known to be present in this tissue. Finally, experiments involving the use of fractions of isolated horizontal cells indicate that these neurons possess a population of VIP receptors coupled to cyclic AMP production which would appear to share a common pool of adenylate cyclase with a population of similarly coupled dopamine receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Down-Regulation of Pinealocyte Protein Kinase C: Effect on α1-Adrenergic Potentiation of β-Adrenoceptor Stimulation of Cyclic AMP Accumulation and Induction of Serotonin N-Acetyltransferase Activity

Treatment of rat pinealocytes with 4 beta-phorbol 12,13-dibutyrate down-regulated protein kinase C (PKC) activity. Loss of activity was concentration-dependent (50% loss at 8 x 10(-7) M after 18 h of treatment) and time-dependent (50% loss after 2 h with 3 x 10(-6) M). Phenylephrine, an alpha 1-adrenergic agonist, and phorbol esters unable to activate PKC did not down-regulate the enzyme. alpha 1-Adrenergic amplification of beta-adrenergic stimulation of cyclic AMP accumulation, a response previously shown to be mediated by PKC activation, was reduced by only 50% in cells in which PKC activity was down-regulated by approximately 95%. These data suggest that there is not a simple proportional relationship between the degree of activation of pinealocyte PKC and the alpha 1-adrenergic amplification of beta-adrenergic cyclic AMP synthesis. In down-regulated cells, alpha 1-adrenergic amplification of beta-adrenergic induction of serotonin N-acetyltransferase activity, a key cyclic AMP-responsive enzyme involved in the nocturnal synthesis of the pineal hormone melatonin, was unchanged. Thus, even though alpha 1-adrenergic amplification of cyclic AMP synthesis is impaired, sufficient cyclic AMP is generated to allow a full induction of serotonin N-acetyltransferase activity. This finding raises the important question of whether the alpha 1-adrenergic amplification mechanism has a physiological role in regulating melatonin synthesis in vivo.     

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.     

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.     

Cholera Toxin Induces Cyclic AMP-Independent Down-Regulation of Gsα and Sensitization of α2-Autoreceptors in Chick Sympathetic Neurons

Abstract: The role of the stimulatory GTP-binding protein (G_S) in the α₂-autoinhibitory modulation of noradrenaline release was investigated in cultured chick sympathetic neurons. The α₂-adrenoceptor agonist UK 14,304 caused a concentration-dependent reduction of electrically evoked [³H]noradrenaline release with half-maximal effects at 14.0 ± 5.5 nM. In neurons treated with 100 ng/ml cholera toxin for 24 h, the half-maximal concentration was lowered to 3.2 ± 1.4 nM without changes in the maximal effect of UK 14,304. The pretreatment with cholera toxin also increased the inhibitory action of 10 nM UK 14,304 when compared with the inhibition of noradrenaline release in untreated cultures derived from the same cell population. In cultures treated with either 10 µM forskolin or 100 µM 8-bromo-cyclic AMP, neither the half-maximal concentration nor the maximal effect of UK 14,304 was altered. Cholera toxin, forskolin, and 8-bromo-cyclic AMP all induced an increase in spontaneous outflow and a reduction in electrically evoked overflow, effects not observed after a pretreatment with dideoxyforskolin. Exposure of neurons to cholera toxin, but not to forskolin or 8-bromo-cyclic AMP, induced a translocation of α-subunits of G_s (G_sα) from particulate to soluble fractions and led ultimately to a complete loss of G_sα from the neurons. In contrast, no effect was seen on the distribution of either α-subunits of G_i- or G_o-type G proteins or of β-subunits. These results indicate that cholera toxin causes a selective, cyclic AMP-independent down-regulation of G_sα. This down-regulation of G_sα is associated with the sensitization of α₂-autoreceptors.     

Studies on the Role of α2-Adrenoceptors in the Control of Synaptosomal [3H]5-Hydroxytryptamine Release: Effects of Antidepressant Drugs

The sensitivity of alpha 2-adrenoceptors on 5-hydroxytryptamine (5-HT) nerve endings obtained from rat cerebral cortex was investigated following treatment with the antidepressant drugs desipramine (10 mg/kg/day for 21-28 days) or clorgyline (1 mg/kg/day for 21-28 days). [3H]5-HT (100 nM) was used to load cortical synaptosomes (P2) after experiments with uptake inhibitors confirmed that this concentration of amine ensured exclusive uptake into 5-HT nerve terminals. The sensitivity of K+-stimulated release of [3H]5-HT to alpha 2-adrenoceptor occupancy was assessed in a superfusion system by means of the dose-dependent inhibition of [3H]5-HT release by clonidine. This is blocked by yohimbine (1 microM), which, when administered alone, enhances release, suggesting that endogenous catecholamines released from other synaptosomes act on these alpha 2-heteroreceptors. The effect of addition of citalopram (1 microM) to superfusates suggests that some reuptake of [3H]5-HT occurs during superfusion. Of the tritium released into superfusates during "background" and K+-stimulated release, 17 and 90%, respectively is [3H]5-HT. The attenuation of K+-stimulated release by clonidine is apparently diminished by the chronic clorgyline regimen but not by desipramine. However, clorgyline elevates catecholamine levels, and this might increase endogenous noradrenaline (NA) efflux, which by competition with clonidine could appear to alter alpha 2-adrenoceptor sensitivity. This possibility was investigated by depleting NA with the neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4). These studies showed that the apparent effect of chronic clorgyline on alpha 2-adrenoceptor sensitivity to clonidine was due to competition with increased levels of endogenous NA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endogenous Noradrenaline Activates α2-Adrenoceptors on Serotonergic Nerve Endings in Human and Rat Neocortex

Abstract: Slices from human neocortex preincubated with [³H]serotonin ([³H]5-HT) were superfused and stimulated electrically to investigate whether the α₂-adrenoceptors on serotonergic terminals can be stimulated by endogenous noradrenaline (NA) released from neighboring noradrenergic fibers. The stimulation-evoked ³H overflow, representing action potential-induced, exocytotic release of 5-HT, was depressed by the NA uptake blocker (+)-oxaprotiline. Rauwolscine (a mixed α₂-adrenoceptor antagonist/5-HT autoreceptor agonist) or phentolamine [a combined α- adrenoceptor/5-HT autoreceptor antagonist; the latter drug in the presence of (+)-oxaprotiline] enhanced the release when the 5-HT autoreceptors had previously been blocked by metitepine. Under hypothermia the release of 5-HT was found to be decreased and that of NA to be increased; under these conditions idazoxan (an α₂-adrenoceptor antagonist) enhanced the release of 5-HT. In neocortex slices from rats (+)-oxaprotiline similarly depressed the release of 5-HT (measured with the same methods) as in human tissue. When rats were pretreated with 6-hydroxydopamine, the inhibitory effect of exogenous NA on 5-HT release was increased, and in slices from rats pretreated with desipramine, it was decreased. In conclusion, α₂-heteroreceptors can be activated by endogenous NA released from neighboring noradrenergic fibers. Because regulatory processes analogous to those in rats probably occur in humans as well, an up- or down-regulation of α₂- heteroreceptors in depressed patients with a (pathological) decrease or a (therapeutic) enhancement of the noradrenergic neurotransmission may also be assumed to occur.