The expression of a cloned Drosophila octopamine/tyramine receptor in Xenopus oocytes

The expression of a cloned Drosophila octopamine/tyramine receptor (OctyR99AB) is described in Xenopus oocytes. Agonist stimulation of OctyR99AB receptors increased intracellular Ca²⁺ levels monitored as changes in the endogenous inward Ca²⁺-dependent chloride current. The receptor is preferentially sensitive to biogenic amines with a single hydroxyl on the aromatic ring. The G-protein, G_αi, appears to be involved in the coupling of the receptor to the production of intracellular calcium signals, since the effect is pertussis-toxin sensitive and is blocked or substantially reduced in antisense knockout experiments using oligonucleotides directed against G_αi but not by those directed against G_αo, G_αq and G_α11. The increase in intracellular calcium levels induced by activation of the OctyR99AB receptor can potentiate the ability of activation of a co-expressed β₂-adrenergic receptor to increase oocyte cyclic AMP levels. A comparison of the pharmacological coupling of OctyR99AB to different second messenger systems when expressed in Xenopus oocytes with previous studies on the expression of the receptor in a Chinese hamster ovary cell line suggests that the property of agonist-specific coupling of the receptor to different second messenger systems may be cell-specific, depending upon the G-protein environment of any particular cell type.     

Regulation of Melatonin Release and N-Acetyltransferase Activity in Ovine Pineal Cells

A suspension culture of ovine pineal cells was developed to investigate the regulation of melatonin synthesis and release. Dosedependent stimulation of melatonin release by a series of adrenergic agonists yielded a typical β-adrenergic profile. Serotonin N-acetyltransferase (NAT), the rate-limiting enzyme in melatonin biosynthesis, was also stimulated by a β-adrenergic mechanism. However, NAT activity appeared less sensitive than melatonin release to β-adrenergic stimulation. No evidence was obtained for a contribution of α₁-adrenergic receptors to the regulation of NAT activity and melatonin release. Activation of adenylate cyclase by forskolin or addition of a cyclic AMP analogue increased both melatonin release and NAT activity. In contrast, the Ca²⁺ ionophore A₂₃₁₈₇ stimulated melatonin release without a detectable increase in NAT activity. Together, the present data argue for a β-adrenergic regulation of both NAT activity and melatonin release in ovine pinealocytes. The evidence also suggests that two intracellular mechanisms may control melatonin release in ovine pinealocytes: a cyclic AMP-dependent mechanism, associated with an increase in NAT activity and a Ca²⁺-dependent mechanism, independent of NAT activity.     

α2-Adrenergic receptors mediate inhibition of cyclic AMP production in neurons in primary culture

The actions of adrenergic agents on the intracellular production of cyclic adenosine monophosphate (AMP) was examined in intact cortical and striatal neurons in primary culture, generated from the fetal mouse brain. Exposure of striatal neurons to the β-adrenergic agonist isoproterenol (10 μM) resulted in a 5-fold increase in intraneuronal cyclic AMP; norepinephrine (100 μM), alone or in combination with isoproterenol, produced only a 3-fold increase in cyclic AMP levels. However, in the presence of yohimbine (10 μM), cyclic AMP productions due to norepinephrine or isoproterenol plus norepinephrine were identical to isoproterenol alone. When striatal or cortical neurons were exposed to pertussis toxin (100 ng/ml) overnight, there was no detectable difference between isoproterenol- and norepinephrine-stimulated cyclic AMP production. These data suggest thatα₂-adrenergic receptors mediate the attenuation of cyclic AMP production in neurons and do so via the inhibitory guanine nucleotide regulatory protein of adenylate cyclase.     

The resolution of dopamine and β1- and β2-adrenergic-sensitive adenylate cyclase activities in homogenates of cat cerebellum, hippocampus and cerebral cortex

The stimulation of adenylate cyclase by dopamine and various β-adrenergic agonists has been investigated in homogenates from 3 areas of cat brain: the cerebral cortex, cerebellum and hippocampus. The purpose of the study was to determine whether the β-adrenergic receptors coupled to adenylate cyclase could be classified as either β₁ and β₂ subtypes in the different regions studied.The stimulation of adenylate cyclase by the β-adrenergic agonist, (−)isoproterenol (5 × 10⁻⁶M), was completely blocked by the specific β-adrenergic antagonist, (−)alprenolol (10⁻⁵ M), but not by the dopaminergic antagonist, fluphenazine (10⁻⁵ M), whereas the stimulation of adenylate cyclase by (−)epinephrine (10⁻⁴ M) was blocked to varying extents by these two drugs in each of the 3 regions studied. The (−)epinephrine effect was always blocked in the combined presence of (−)alprenolol and fluphenazine. The adenylate cyclase stimulation by (−)epinephrine which is not blocked by (−)alprenolol was due to interaction of (−)epinephrine with a dopaminergic-sensitive adenylate cyclase which has been characterized in cerebral cortex, hippocampus and cerebellum.Regional differences in the affinity of β-adrenergic-sensitive adenylate cyclase for various agonists were investigated in the presence of fluphenazine (10⁻⁵ M). In the cerebellum the potency order was (±)protokylol> (±)hydroxybenzylisoproterenol> (±)isoproterenol> (−)epinephrine> (±)salbutamol> (−)norepinephrine, indicating the presence of a β₂-adrenergic receptor. In the cerebral cortex the potency order was (−)isoproterenol> (±)protokylol> (±)hydroxybenzylisoproterenol> (−)epinephrine= (−)norepinephrine((±)salbutamol being inactive). A similar pattern was found in the hippocampus indicating the presence of a β₁-adrenergic receptor in these two regions. (±)Salbutamol was a partial agonist in the cerebellum and a competitive antagonist in the cerebral cortex.The ratio of the antagonist potencies of (±)practolol and (±)butoxamine preferential β₁- and β₂-adrenergic antagonists respectively, to block the stimulation of adenylate cyclase was 25 in the cerebellum, compared to 0.5 in the cerebral cortex and 1.6 in the hippocampus. These results confirm the presence of a β₂ subtype of receptor coupled to adenylate cyclase in the former and β₁ subtypes in the latter two regions. The comparison between the affinities of a series of β-adrenergic agonists and antagonists for the β-adrenergic receptors coupled with an adenylate cyclase in cerebral cortex and cerebellum with their affinities for well characterized β₂-adrenergic receptors in lung and β₁-adrenergic receptor in heart substantiated this conclusion.     

Regulation of central α-adrenoceptors by serotoninergic denervation

α₁- and α₂-adrenoceptors were assessed by binding studies using [³H]prazosin and [³H]p-aminoclonidine as ligands in membrane preparations from the cortex, hippocampus and hypothalamus of rats, 3 weeks after intracerebroventricular injection of the neurotoxin 5,7-dihydroxytryptamine. Cortical α₁ and hippocampal α₂ adrenoceptors were significantly increased. Treatment also affected the affinity of cortical α₂ adrenoceptors. These results suggest a heterologous, region-specific regulation of both subtypes of central α-adrenergic receptors by serotonin.     

Morphine dependence in human neuroblastoma SH-SY5Y cells is associated with adaptive changes in both the quantity and functional interaction of PGE1 receptors and stimulatory G proteins

Chronic exposure of all-trans-retinoic acid-differentiated SH-SY5Y cells to morphine (10 μM; 2 days) results in sensitization of adenylate cyclase as characterized by a significant increase in both PGE1 receptor-mediated as well as receptor-independent (NaF, 10 mM; forskolin, 100 μM) stimulation of effector activity. To investigate the underlying biochemical alterations, chronic opioid regulation of each of the components comprising the stimulatory PGE, receptor system was examined. On receptor level, chronic morphine treatment was found to reduce PGE₁ receptor number (B_max) by approximately 40%, whereas their affinity slightly increased. Binding experiments performed in the presence of GTPγS (100 μM) further indicate that the decrease in PGE1 receptor density is associated with a loss of functionally G protein-coupled receptors. On post-receptor level, chronic morphine treatment substantially increased the abundance and functional activity of stimulatory G proteins, as assessed by cholera toxin-catalyzed ADP-ribosylation of G_sα and S49 cyc⁻ reconstitution assays. No changes were found on the level of adenylate cyclase. Evaluation of the functional interaction between PGE₁ receptors and G_s in situ by application of a C-terminal anti-G_sα antibody revealed a more intense coupling efficiency between these two entities, since a significant higher amount of antibody (2.3-fold) was required in morphine dependent cell membranes to half-maximally attenuate PGE₁ receptor-stimulated adenylate cyclase activity. In addition, limitation of the amount of functionally available G_sα within the PGE₁ receptor/adenylate cyclase signal transduction cascade abolished the generation of a supersensitive adenylate cyclase response during the state of naloxone (100 μM)-precipitated withdrawal. These data demonstrate that in human neuroblastoma SH-SY5Y cells chronic morphine-induced sensitization of adenylate cyclase is associated with distinct quantitative and qualitative adaptations within the stimulatory adenylate cyclase-coupled PGE₁ receptor system. Thus, alterations in the functional activity of stimulatory receptor systems are suggested to contribute to the cellular mechanisms underlying opioid dependence.     

ATP increases extracellular dopamine level through stimulation of P2Y purinoceptors in the rat striatum

The effect of ATP on release of dopamine (DA) from rat striatum was studied using in vivo microdialysis. ATP increased the striatal extracellular levels of DA dose-dependently. These analogs produced an increase in DA according to this order of potency: 2-methylthio ATP > ATP α, β-methylene ATP > ADP > AMP > adenosine. Adenosine 5′-[β, γ imido]-triphosphate had a more prolonged effect on the increase in DA level than ATP. The ATP-induced increase in DA was inhibited by adding suramin, a nonselective P₂ purinoceptor antagonist, and reactive blue 2, a P_2Y purinoceptor antagonist, but not inhibited by xanthine amine congener, an adenosine receptor antagonist. Pertussis toxin reduced the increase in DA produced by ATP, whih suggests that te P₂ purinoceptor may be coupled with a G-protein in the rat striatum. Results suggest that P_2Y purinoceptors may involve an ATP-induced increase in DA. The ATP-induced release of DA was tetrodotoxin-sensitive, Ca²⁺-dependent and was abolished by ω-conotoxin GVIA, indicating that the opening of voltage-sensitive Na⁺ channel and the Ca²⁺ influx through the N-type voltage-dependent calcium channel are both required for the ATP-induced increase in DA. The ATP-induced increase in DA is presumably due to the release of DA via the stimulation of P_2Y purinoceptors in the rat striatum.     

Characteristics of the β1- and β2-adrenergic-sensitive adenylate cyclases in glial cell primary cultures and their comparison with β2-adrenergic-sensitive adenylate cyclase of meningeal cells

The agonist specificity pattern of the β-adrenergic adenylate cyclase in glial primary cultures was not typical of either β₁- or β₂-adrenergic receptors. The dose-response curves for adrenaline did not correspond to simple mass action kinetics and their computer analysis suggests the presence of both β₁- and β₂-adrenergic-sensitive adenylate cyclase (58 ± 17% and 42 ± 17% respectively).Similar properties of β₁- and β₂-adrenergic-sensitive adenylate cyclases were found by computer analysis of the dose-response curves for isoprenaline in the presence of a constant concentration of practolol (a selective β₁ antagonist) ( 55 ± 10% and 45 ± 10% of β₁- and β₂-sensitive adenylate cyclase respectively).The curves for displacement of [³H]dihydroalprenolol by practolol confirm these results.For purpose of comparison, the β-adrenergic receptors of meningeal cells in cultures were subjected to similar analysis. The results clearly showed that these cells exclusively contained β₂-adrenergic receptors.     

Corticosterone alters G protein α-subunit levels in the rat hippocampus

The hypothalamic-pituitary-adrenal axis regulates the synthesis and secretion of corticosteroid hormones. The hippocampus, a component of the limbic system, contains the highest concentration of corticosteroid receptors in the brain and may play an important role in regulating hypothalamic-pituitary-adrenal axis activity and mediating physiological responses to stress. The corticosteroid hormone corticosterone alters the response elicited by activation of several different G protein-linked neurotransmitter receptors in the hippocampus. In the present study we used Western blot and immunohistochemical techniques to determine the effects of chronic adrenalectomy (ADX), low basal (CT) and high (HCT) corticosterone treatments on G_s, G_{i1 and 2} and G_o α-subunit levels and intracellular location in the rat hippocampus. CT treatment increased G_s α-subunit levels and HCT treatment increased the levels of G_s, G_{i1 and 2} and G_o α-subunits when compared to sham as detected on Western blots. No change in the intracellular location of the G protein α-subunits was detected using immunohistochemistry. Based on our results, we conclude that corticosterone alters G protein α-subunit levels in the rat hippocampus without altering their intracellular location. These results provide an important piece of information towards understanding how corticosteroids alter G protein-linked neurotransmitter receptor-mediated responses.     

Interaction betweenα2- and β-adrenergic receptors in rat cerebral cortical membranes: clonidine-induced reduction in agonist and antagonist affinity for β-adrenergic receptors

The interaction betweenα₂- and β-adrenergic receptors was investigated in rat cerebral cortical membranes. Clonidine inhibition of [³H]dihydroalprenolol ([³H]DHA) binding resulted in biphasic competition curves with a mean Hill coefficient of 0.45. The addition of 1 μM yohimbine caused a rightward shift of the first portion of the clonidine inhibition curve. In the presence of 1 μM clonidine, the maximum concentration which did not inhibit [³H]DHA binding, inhibition curves of [³H]DHA binding by isoproterenol shifted to the right. A mean Hill coefficient increased from a control value of 0.63 to 0.76. Computer modeling analysis revealed that 1 μM clonidine decreased a β-adrenergic high-affinity state from 28% to 13%. However, the addition of 1 μM yohimbine completely prevented the clonidine-induced reduction in the β-adrenergic high-affinity state. In the presence of 200 μM GTP, the effect of clonidine was not observed. In addition,K_d andB_max values for[³H]p-aminoclonidine ([³H]PAC) binding were not significantly changed by the addition of 100 nM isoproterenol, the maximum concentration which did not inhibit [³H]PAC binding. Moreover, isoproterenol inhibition of [³H]PAC binding resulted in steep competition curves with a mean Hill coefficient of 0.97. The addition of 1 μM alprenolol did not affect the isoproterenol inhibition curve. These data demonstrated that clonidine caused a decrease in agonist and antagonist affinity for β-adrenergic receptors, while isoproterenol did not modulate the binding characteristics ofα₂-adrenergic receptors. Furthermore, these results suggest that regulation betweenα₂- and β-adrenergic receptors is not bidirectional, but is instead unidirectional fromα₂-adrenergic receptors to β-adrenergic receptors.     

Vasoactive intestinal peptide stimulates melatonin release from perifused pineal glands of rats

Summary The rat pineal gland is known to release melatonin in response to noradrenergic stimulation. The effect of vasoactive intestinal peptide (VIP), one of the neuropeptides present in the pineal, was examined on perifused rat pineal glands. VIP stimulated melatonin release with a dose-dependent effect above 10^–7 M. In regard of kinetic characteristics, the pattern of melatonin release after VIP stimulation was similar to that after isoproterenol stimulation. 10^–6 M VIP-stimulated melatonin release was not altered when the pineal glands were treated with 10^–5 M propranolol (a -adrenergic antagonist) or 10^–5 M prazosin (an ₁-adrenergic antagonist). Thus VIP has a noradrenergic-independent effect on melatonin secretion. Conversely, this VIP effect is greatly inhibited by the specific action of a VIPergic antagonist. This suggests that VIP acts on melatonin synthesis through its own binding sites.This study demonstrates that melatonin secretion from rat pineal glands may be elicited through a VIPergic system which is independent of the well-known noradrenergic system.     

Catecholaminergic regulation of proliferation and survival in rat forebrain paraventricular germinal cells

We have investigated the possible role of α₁-adrenoreceptors in regulating the germination of progenitor cells cultured from embryonic rat neocortex. High binding levels of the α₁-selective radioligand ³[H]prazosin were detected in the forebrain of the rat embryo at E13, and the greatest density of binding sites was localized to the ventricular and subventricular zones. Catecholamine-containing axon terminals were present in these zones in the same period. Germinal neuroepithelial cells retained specific ³[H]prazosin binding in culture. ≈25% of cells in culture displayed complex intracellular Ca²⁺ transients in response to phenylephrine, many of which were abolished with the α_1B antagonist, chloroethylclonidine. Cultures exhibited concentration-dependent catecholamine stimulation of DNA synthesis mediated by α₁ receptors in serum-limited conditions. Neuroepithelial cells were labelled via their ventricular processes by intraventrivcular injection of Fast blue in E13 embryos prior to transfer of the neocortex to dissociated cell culture. Many of labelled cells were present in culture in germinal foci. Some cells which migrated from these foci underwent apoptosis, as determined by TUNEL in situ hybridization. During a transitory period of up to 48 h in culture, α₁-adrenoreceptor activation by phenylephrine or noradrenaline increased the number of surviving cells. Apoptosis was observed in vivo in both ventricular and subventricular zones of the neocortex from E13 to E15 in increasing numbers. We propose that both the supply of noradrenaline to forebrain germinal cells, and the expression of α₁-adrenoreceptors on their surface could act to determine whether they die or continue to proliferate.     

Pineal response to isoproterenol in rats chronically treated with electroconvulsive shock

Chronic electroconvulsive shock (ECS) has been shown to induce a downregulation of β₁-adrenergic receptors in the rat cerebral cortex. Because the secretion of melatonin in the pineal gland is regulated primarily by β₁-adrenoceptors, in the present study we investigated the effect of chronic administration of ECS on pineal β-adrenergic responsiveness to isoproterenol. To this purpose, young adult male rats received once daily for 8 days ECS (80 mA 0.5 s) or sham ECS. On the day after the last ECS or sham treatment, they were injected with isoproterenol hydrochloride (1 mg/kg SC) or volume-matched saline at 1600 h. Two hours later they were killed by decapitation. Results showed that the isoproterenol-induced increase in the pineal melatonin content was blunted in rats treated with ECS as compared to sham-treated animals (shock × druginteraction = p < 0.01). These data indicate that chronic ECS treatment affects β₁ receptor-mediated melatonin production in the pineal gland. Further studies need to elucidate whether the blunted melatonin response to isoproterenol in ECS-treated rats is due to a downregulation of pinealocyte β-adrenergic receptors.     

Light-induced decrease of serotonin N-acetyltransferase activity and melatonin in the chicken pineal gland and retina

In the pineal gland and retina of chickens, light exposure at night when serotonin N-acetyltransferase (NAT) activity levels are high causes a 4–5-fold decrease in NAT activity. Thet_1/2 of NAT inactivation is 10 min and the kinetics of inactivation are similar in the pineal gland and retina. A brief pulse of light can initiate the process of NAT inactivation which continues in the dark for 30 min before a partial recovery of NAT activity occurs. In blinded chicks, there is less inactivation of pineal NAT by light than in intact chicks, indicating that the eyes are involved in the pineal's response to light. In chicks that have had their superior cervical ganglia removed inactivation of pineal NAT by light is intermediate between that of intact and blind chicks, indicating that ganglionectomy does not completely mimic the effects of blinding.The pineal gland itself is light-sensitive in culture. Light causes a 4–5-fold decrease in NAT activity in static organ culture, and inhibits melatonin release in flow-through organ culture. Drugs that increase cyclic nucleotide levels in cells (cholera toxin, RO 20-1724, monobutyryl cyclic AMP, monobutyryl cyclic GMP) block the NAT decrease by light, whereas high potassium or EGTA do not block this light-induced NAT inactivation.     

Distribution of α-adrenergic, β-adrenergic and dopaminergic receptors in discrete hypothalamic areas of rat

Catecholamine receptor binding sites were measured in discrete hypothalamic nuclei or regions as well as in certain extrahypothalamic areas of the adult male rat. For each assay, discrete areas were microdissected from frozen tissue sections and pooled from several animals. Specific high affinity binding sites were assessed at fixed ligand concentrations for [³H]p-aminoclonidine (PAC) and [³H](2-C 2′,6′-(CH₃O)₂ phenoxyethylamino)-methylbenzodioxan (WB-4101) for α-adrenergic receptor sites, for [³H]dihydroalprenolol (DHA) for β-adrenergic receptor sites, and for [³H]2-amino-6, 7-dihydroxy-1,2,3,4-tetrahydronaphtalene (ADTN) and [³H]spiroperidol in the presence of cinanserin for dopaminergic receptor sites.Regional variations in [³H]WB-4101 binding were relatively small in magnitude, with most hypothalamic and extrahypothalamic areas possessing between 60 and 90% of the binding in frontal cortex. [³H]PAC binding showed a wider range of binding density across brain areas than did [³H]WB-4101, but, in general, variations in [³H]PAC binding paralleled those in [³H]WB-4101 binding. In hypothalamus, binding was characterized as being predominantly to α₁-receptors in the of [³H]WB-4101 and to α₂-receptors in the case of [³H]PAC. The medial hypothalamic areas exhibited a somewhat higher density of these α-adrenergic sites than did the lateral hypothalamus (perifornical hypothalamus and medial forebrain bundle). Also, the ratio of [³H]PAC to [³H]WB-4101 binding differed in different hypothalamic areas, ranging from 1.5:1 to 4:1. The median eminence was exceptional in that it contained appreciable [³H]PAC but no significant [³H]WB-4101 binding sites at the radioligand concentrations tested. Binding of [³H]DHA to β-adrenergic receptors varied over approximately a 3-fold range in the different hypothalamic areas, with binding highest in the medial forebrain bundle and the medial preoptic area, and lowest in the periventricular, dorsomedial and posterior hypothalamic nuclei, the median eminence and the zona incerta. The ratio of β-adrenergic to α-adrenergic binding sites was generally lower in the medial than in the lateral hypothalamic areas and higher in the extrahypothalamic areas examined than in the hypothalamus. With regard to [³H]spiroperidol and [³H]ADTN binding to dopaminergic sites, the striatum, nucleus accumbens and olfactory tubercle showed a greater density of [³H]spiroperidol than of [³H]ADTN sites, in contrast to the hypothalamus where [³H]ADTN binding was more predominant. Within the hypothalamus, [³H]ADTN binding was relatively uniform, while [³H]spiroperidol binding was quite high in four hypothalamic areas (lateral perifornical area, medial forebrain bundle, paraventricular and dorsomedial nuclei), intermediate in the median eminence and arcuate nucleus, and low or not detectable in all other hypothalamic areas.     

Prostaglandin E2 mediates the stimulatory effect of methoxamine on in vivo luteinizing hormone-releasing hormone (LH-RH) release in the ovariectomized female rhesus monkey

Previously, we found that noradrenergic input throughα₁-receptors modulates pulsatile release of luteinizing hormone-releasing hormone (LH-RH) in ovariectomized rhesus monkeys in the absence of estrogen. In the present study, the role of prostaglandin E₂ (PGE₂) in mediating α-adrenergic stimulation of LH-RH release is investigated. In the first experiment the effects of theα₁-adrenergic agonists methoxamine (MTX) on LH-RH and PGE₂ release were examined. Push-pull perfusion of the stalk-median eminence (S-ME) was performed in conscious, ovariectomized monkeys, and perfusate samples were collected on ice. MTX (10⁻⁵ M) was infused into the S-ME through the push cannula for 10 min at 90-min intervals, and LH-RH and PGE₂ in aliquots of the same perfusate samples were measured by radioimmunoassay. Infusion of MTX significantly stimulated LH-RH release (n = 12; P < 0.01) and PGE₂ release (P < 0.05). In the second experiment, the effect of PGE₂ infusion on LH-RH release was tested. PGE₂ (10⁻⁷ M) was infused using the same protocol as above, and LH-RH was measured in the perfusates. Infusion of PGE₂ through the push cannula significantly stimulated LH-RH release (n = 23; P < 0.05). These results suggest that the stimulatory effect of MTX on LH-RH release is at least partly mediated by PGE₂, since MTX stimulated not only LH-RH but also PGE₂ release, and since PGE₂ itself stimulated LH-RH release. Therefore, PGE₂ may be an important endogenous mediator ofα₁-adrenergic input stimulating pulsatile PH-RH release. Moreover, the stimulatory effects of MTX and PGE₂ can be observed in the absence of estrogen in the rhesus monkey, unlike in rhodents. Our results also demonstrate the usefulness of the push-pull perfusion technique for studies of cellular mechanisms in neuroendocrine research.     

Development of the rat pineal #x003B1;1-adrenoceptor

Pineal α₁-adrenoceptors in rats from 19 days of gestation until 11 months of age were studied using [¹²⁵I]iodo-2-[β-(4-hydroxyphenyl)ethylaminomethyl]tetralone ([¹²⁵I]HEAT). The number of specific [¹²⁵I]HEAT binding sites increased markedly between 18 days of gestation (101.7 ± 13.1fmol/mg protein) and 10 days of age (336.2 ± 34.3fmol/mg protein). A significant decline occurred after 1 month of age. A saturation study showed similar change in receptor density with age (B_max; 20 days of gestation, 130.5 fmol/mg protein; 35 days old, 288.1 fmol/mg protein) but no difference in K_d(58.4pM at both− 1and+ 35days). The developmental appearance of the pineal α₁-adrenoceptor and the decline in its density with age are remarkably similar to changes reported for pineal β-adrenoceptors.     

Behavior and binding: Correlations between α1-adrenergic stimulation of acoustic startle and α1-adrenoceptor occupancy and number in rat lumbar spinal cord

The relationship between alterations in α₁-adrenoceptors and behavioral effects of α₁-adrenergic agonists were investigated in a localized region of the rat central nervous system. Direct infusion of the α₁-adrenergic agonists,d-amphetamine or phenylephrine. into the subarachnoid space of the lumbar cord (intrathecal administration) increased the amplitude of the acoustic startle reflex, The magnitude of this behavioral facilitation correlated highly with the degree of α₁-adrenoceptor occupation measured by [³H]prazosin binding in lumbar spinal tissue. Using an in vitro estimate of receptor occupation, maximal potentiation of startle occurred following approximately 30% occupation of the receptors, using eitherd-amphetamine or phenylephrine. Intrathecal administration of 6-OHDA produced a 95% decrease in spinal norepinephrine and markedly enhanced the behavioral response to intrathecal phenylephrine as well as the number of α₁-adrenoceptors. The correlation between the time course of the behavioral and binding changes was 0.99. No change in receptor affinity (K_D) or receptor occupation by phenylephrine was found after 6-OHDA. The data indicate that receptor binding parameters do have predictive value for behavior, especially if localized regions of the nervous system, critical to the behavior, are analyzed.     

Epinephrine release and presynaptic α2-adrenoceptic regulation in the guinea pig hypothalamus

The release of epinephrine (E) and its regulation system were investigated in the slices of guinea pig hypothalamus. Electrical stimulation produced an efflux of [³H]E from tissues preloaded with [³H]E, in a current- and frequency-dependent manner. Stimulated [³H]E release was inhibited by tetrodototxin and by a calcium-free medium containing ethylene glycol bis(gb-aminoethylether)-N,N,N′ N′-tetraactive acid (EGTA). Stimulated [³H]E release was enhanced by yohimbine and this effect was suppressed by clonidine. These results provide strong evidence for the neurotransmitter role of E inthe hypothalamus, and suggest the possible existence of regulatory mechanism of E release via presynaptic α₂-receptors.