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.     

The interaction between β- and α2-adrenoceptors in cerebral cortical membranes: Isoproterenol-induced increase in [H]clonidine binding in rats

The pretreatment of rat cerebral cortical membranes with 10 or 100 μM isoproterenol at 37°C for 40 min caused a significant elevation of the B_max value of [³H]clonidine binding but pretreatment at 4°C did not affect the value. The isoproterenol-induced increase in the B_max value of the binding was higher in 50 mM Tris-HCl buffer (pH 7.7) than in Krebs-Ringer solution. In 50 mM Tris-HCl buffer (pH 7.7), treatment with isoproterenol reduced the B_max value of [³H]dihydroalprenolol binding but neitherK_d nor B_max of [³H]WB 4101 binding was affected by this treatment. Fitty μM propranolol or 100 μM GTP produced a significant reduction in isoproterenol-induced elevation of the B_max value of [³H]clonidine binding. In contrast, 100 μM cyclic AMP did not affect the control binding and 0.1 or 1 mM theophylline did not affect the isoproterenol-induced elevation of the binding. The only B_max value in high affinity binding of [³H]clonidine was increased by isoproterenol.It is suggested that isoproterenol increases the density of α₂-adrenoceptors in a temperature-dependent manner. The direct interaction between β- and α₂-receptor molecules and/or their indirect interaction, mediated by GTP regulatory proteins, would exist in the cerebral cortical membranes of rats.     

Characterization of β-adrenergic receptors in rat brain and pituitary using a new high-affinity ligand, [I]iodocyanopindolol

The binding of [¹²⁵I]iodocyanopindolol (ICYP) to membrane preparations from rat cerebral cortex, hypothalamus and anterior pituitary gland was characterized in regard to specificity, density, and the proportion of β-adrenergic receptor subtypes. By employing a mixture of ligands specific for α-adrenergic, serotoninergic and dopaminergic receptors, it was possible to eliminate most of the less-specific contributions to ICYP binding profiles, which resulted in narrowing the range of measured dissociation constants to 35–50 pM for all neural tissues studied. These values corresponded well with constant for the ‘slow’ component discernible in ICYP association with cerebral cortical membranes at 37° C. The maximum binding values were 63, 29 and 5.6 fM/mg membrane protein in cortical, hypothalamic and anterior pituitary membrane fractions, respectively.Evaluation of the β-adrenergic receptor subtypes using 4 selective competitors indicated an average 19% content of the β₂-subtype in cortical membranes, while in hypothalamic membranes 47% of the receptors could be assigned to that subtype. In the anterior pituitary as well as in the cerebellum, the receptors were predominantly ofβ₂-subtype. These findings are discussed in terms of possible physiological functions of β-receptors in these tissues, including the regulation of the release of pituitary hormones.     

Characterization of [I]HEAT binding to α1-receptors in human brain: assessment in aging and Alzheimer's disease

The binding of [¹²⁵I]2-(β-4-hydroxyphenylethylamino-ethyltetralone ([¹²⁵I]HEAT), an α₁-adrenergic receptor antagonist, to human brain membranes was characterized and the binding assessed in tissue from subjects with Alzheimer's disease (AD) and aging controls. Under Na⁺-K⁺ phosphate buffer conditions, [¹²⁵I]HEAT bound to a single class of binding sites in prefrontal cortex (Brodmann area 10) with a K_d of about 120 pM. High binding capacities of [¹²⁵I]HEAT were evident in the hippocampus and neocortex but were low in subcortical areas and cerebral microvessels comparable to the regional distribution of [³H]prazosin binding reported previously. Displacement of [¹²⁵I]HEAT by various adrenergic drugs was consistent with its binding to α₁-adrenergic receptors. The specific binding was not affected by postmortem delay between death and freezing of tissue at autopsy. There was no correlation of [¹²⁵I]HEAT binding with age of subjects. In AD subjects, the binding was significantly decreased in prefrontal cortex by about 25% but not changed in hippocampus, putamen or cerebellum compared to age-matched controls. The reduced binding of [¹²⁵I]HEAT in prefrontal cortex may reflect a region-specific change in α₁-adrenergic receptors associated with neuronal loss in AD.     

Selective increases in the density of cerebellarβ1-adrenergic receptors

Destruction of noradrenergic neurons by 6-hydroxydopamine or chronic blockade of β-adrenergic receptors with propranolol increased the density ofβ₁-adrenergic receptors two-fold in rat cerebellum but had no effect on the density ofβ₂-adrenergic receptors. The results suggest that even thoughβ₁ receptors comprise only 5–10% of the total number of β-adrenergic receptors in the cerebellum they are the receptors specifically innervated by noradrenergic neurons and they may thus be the physiologically important receptors.     

Evidence for central α2-adrenergic modulation of rat pineal melatonin synthesis

This study was performed to distinguish central and peripheral α₂-adrenoceptors in the inhibition of rat pineal melatonin synthesis. The rats received lipo- or hydrophilic α₂-adrenoceptor ligand injections at middark; after 1 or 2 h the pineal melatonin contents were measured. The lipophilic agonist medetomidine (100 μg/kg s.c.) suppressed the melatonin contents significantly, while the hydrophilic agonists ST-91 and p-aminoclonidine (10 or 100 μg/kg i.v.) did not. The suppression by medetomidine was counteracted by the lipophilic antagonist yohimbine (0.3–3.0 mg/kg i.p.) but not by the hydrophilic antagonist L-659,066 (1–10 mg/kg i.v.). In conclusion, the suppression of nocturnal melatonin synthesis by α₂-adrenoceptor agonists is mainly of central origin.     

α2-Adrenergic receptors inhibit catecholamine secretion from bovine adrenal medulla

Catecholamine secretion evoked by carbymlcholine from isolated bovine adrenalinal medullary cells was inhibited byα ₂-agonist, clonidine, in a dose-dependent manner with IC₅₀ value of 2.8 × 10⁻⁵ M. [³H]Clonidine bound to adrenal medullary membranes with high affinity and saturable characteristics. These results suggest thatα ₂-adrenergic receptors which exist on adrenal medullary cells have inhibitory effect on the secretion of catecholamine from the cells     

A comparison of α2-adrenoceptor regulation in brain and platelets

Functional responses andα₂-adrenoceptor radioligand binding were studied in brain and platelets of rabbits under a variety of circumstances. The effects of oestrogen treatment and maturation were studied in female rabbits and of aging and amitriptyline treatment in male rabbits. No correlation was found between changes in brain and platelets either in response orα₂-adrenoceptor ligand binding under any of the conditions examined.     

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.     

Effects of diet and obesity on brain α1- and α2-noradrenergic receptors in the rat

The chronic feeding of a sweetened condensed milk/corn oil diet (CM diet) to adult male rats produced significant increases in body weight and levels of plasma insulin in 34% of the rats fed this diet with respect to chow-fed controls. Levels of alpha 1-noradrenergic receptor binding were lower (32%) in the hypothalamic ventromedial nucleus (VMN) of only those rats which became obese (DIO rats) with respect to both chow-fed controls and those rats which resisted the development of obesity on the CM diet (DR rats). Also, alpha 1-noradrenergic binding was inversely proportional to body weight gain in the VMN (r = -0.831). alpha 2-Noradrenergic receptors were 30-37% lower in both the DIO and DR rats in the dorsomedial nucleus and dorsal area of the hypothalamus, and the medial dorsal area and nucleus reuniens of the thalamus. The similar decreases in alpha 2-noradrenergic receptors in both the DIO and DR rats in these areas suggested that dietary factors alone were responsible for these changes. There were no significant differences from chow-fed rats for hypothalamic dopamine (D2) or beta-noradrenergic (beta 1- and beta 2-) receptors in either DR or DIO rats. These results indicate that VMN alpha 1-noradrenergic receptors co-vary with body weight and implicate a role for alpha 1-receptors in the VMN in the central neuronal regulation of body weight.     

Autoradiographic distribution of α2 adrenoceptors, NAIBS, and 5-HT1A receptors in human brain using [H]idazoxan and [H]rauwolscine

The regional distribution of [³H]idazoxan and [³H]rauwolscine was studied autoradiographically in human brain. [³H]Idazoxan binds with high affinity to α₂ adrenoceptors as well as to non-adrenergic sites (NAIBS). [³H]Rauwolscine, besides binding to α₂ adrenoceptors, also binds to 5-HT_1A receptors. Both radioligands labelled the same population of α₂ adrenoceptors, defined as the epinephrine-displaceable binding component. The highest densities of α₂ adrenoceptors occur in the leptomeninges, cerebral cortex and claustrum; lower densities were visualised in the basal ganglia, thalamus, pons, substantia nigra, cerebellum and medulla oblongata; no α₂ adrenoceptors were detected in amygdala and nucleus ruber. NAIBS were present in all the examined brain areas, with the highest densities found in the basal ganglia and substantia nigra. The finding that certain brain regions, such as the amygdala, contained NAIBS but no detectable α₂ adrenoceptors, suggests that the binding sites are independent from each other. The regional distribution of 5-HT_1A receptors labelled by [³H]rauwolscine is in agreement with previous studies using [³H]8-OH-DPAT.     

Activation of α2-adrenergic receptors decreases nerve trauma-induced afferent barrage but not autotomy

Effects of the selective α₂-adrenoceptor agonist, medetomidine, on a compound volley of a tibial nerve stimulation-evoked spinal reflex, pain-induced phrenic motor responses and on postoperative neuropathic pain behavior were studied in rats. Medetomidine (0.3 mg/kg) decreased the amplitude of the compound volley recorded from peroneal nerve in response to tibial stimulation in pentobarbital (40 mg/kg) anesthetized rats. Atipamezole, an α₂-adnenoceptor antagonist (1.5 mg/kg) fully restored the response when given 60 min after the medetomidine administration. Pain-evoked phrenic motor responses were completely inhibited upon combination anesthesia by pentobarbital (40 mg/kg) and medetomidine (0.3 mg/kg) (PB+M) but not upon plain pentobarbital anesthesia (50 or 60 mg/kg) (PB50, PB60). To study the effect of medetomidine on postoperative neuropathic pain behavior (autonomy), transection of sciatic nerve was done under PB+M, PB50 or PB60 anesthesia. No differences between the groups were found in the postoperative pain behavior during eight-week follow up. The results show that activation of α₂-adrenergic receptors by medetomidine under pentobarbital anesthesia mitigates trauma-induced afferent barrage, whereas it does not reduce the subsequent autotomy.     

Supersensitivity of analgesic responses to α2-adrenergic agonists in genetically hypertensive rats

This study compared the responsivity of α₂-adrenoceptors to agonists in normotensive (WKY) and genetically hypertensive (SH) rats. Clonidine produced a greater degree of analgesia in SH as compared to WKY rats. This analgesia was antagonized by yohimbine. Neither naphazoline nor 4-hydroxy-clonidine produced analgesia in SH or WKY rats. Our results suggest that the analgesic effects of clonidine in SH rats are probably mediated by supersensitive centralv α₂-adrenoceptors.     

Comparison of the binding of nicotinic agonists to receptors from human and rat cerebral cortex and from chick brain (α4β2) transfected into mouse fibroblasts with ion channel activity

(−)-Nicotine, cytisine and carbachol evoked⁸⁶Rb efflux from mouse fibroblasts stably transfected with α₄β₂ chick brain nicotinic subunits. This response to (−)-nicotine was inhibited by mecamylamine and dihydro-β-erythroidine and was mirrored by a rise in intracellular Ca²⁺ measured by microspectrofluorimetry. Lobeline and isoarecolone methiodide evoked no significant⁸⁶Rb from cells and unlike the above agonists displayed significantly different IC₅₀ values for the displacement of [³H]nicotine from mammalian (rat and human cerebral cortex) and transfected fibroblast membranes.     

Autoradiographic localization ofβ1 andα1-adrenoceptors in the midbrain and forebrain of normal and reeler mutant mice

The distribution of alpha-1 and beta-1 adrenoceptors has been studied in the midbrain and forebrain of normal and reeler mutant mice, using autoradiographic visualization of radioiodinated HEAT and ICYP, respectively. All cortical structures and nuclear groups of the murine forebrain and midbrain bind ICYP and HEAT. For each ligand, there is substantial regional variation in binding density and these variations tend to observe boundaries between nuclei or cortical regions or the stratification of cortical regions. Regional variations in binding densities are generally different for ICYP and HEAT. Binding sites for ICYP are distributed densely throughout all fields of the neocortex (particularl, layersI–III > VI) and paleocortex, the striatum, pallidum, substantia nigra and superficial strata of the superior colliculus. Dense concentrations of binding sites for HEAT in cortical structures, by contrast, are limited to frontal (all layers except IV) and anterior cingulate regions of the neocortex and, as with ICYP, the stratum lacunosum-moleculaire of the regio superior of the hippocampal formation. In subcortical structures, again in contrast to the pattern with ICYP, binding density is greatest in the principal nuclei of the dorsal thalamus and the septal nuclei. the regional binding patterns of both ICYP and HEAT in the reeler brain are identical to those in the normal animal. Differential laminar binding patterns within the neocortex are approximately inverted in the two genotypes, however. Thus, binding of ICYP is densest in an inner zone of the mutant, but in the outer 3 layers of the normal neocortex. Binding of this ligand is of relatively lower density in an outer zone of the mutant and in the inner 3 layers of the normal neocortex. Similar inversions are characteristics of the laminar binding patterns of HEAT in the frontal, primary sensory and associational cortical regions of the two genotypes where densest binding is encountered superficially in reeler but at deeper levels of the normal neocortex.     

α2-Adrenoceptors in human forebrain: autoradiographic visualization and biochemical parameters using the agonist [H]UK-14304

The regional distribution and biochemical parameters ofα₂-adrenoceptors in post-mortem human brain tissue were analyzed in autoradiographic and membrane binding studies, using the full agonist, [³H]UK-14304. Autoradiographic visualization of these receptors in the forebrain revealed a heterogeneous anatomical distribution with high levels in the neocortex, ventral hypothalamus, hippocampus and some thalamic nuclei, among others. In membrane binding studies, analyses of saturation curves indicated the presence of a single population of sites for [³H]UK-14304 both in human and rat brain. There was a good correlation between the density of aα₂-adrenoceptors obtained by both procedures. [³H]UK-14304 is a feasible ligand to localise and quantifyα₂-adrenoceptors in human post-mortem material by autoradiographic and membrane binding techniques.     

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.     

α2, α2A, α2B/2C-Adrenoceptor subtype antagonists prevent lipopolysaccharide-induced fever response in rabbits

Exogenous pyrogens, e.g., bacterial lipopolysaccharides (LPS), are thought to stimulate macrophages to release endogenous pyrogens, e.g., TNFα, IL-1 β, and IL-6, which act in the hypothalamus to produce fever. We studied the effect of different α₁⁻ and α₂-adrenoceptor subtype antagonists, applied intraperitoneally, on the febrile response induced by LPS in rabbits. Evidence was obtained that prazosin, an α₁⁻ and α_2B/2C-adrenoceptor antagonist; WB-4101, an α₁⁻ and α_2A-adrenoceptor antagonist; CH-38083, a highly selective α₂-adrenoceptor antagonist (α₂: α₁ > 2000); BRL-44408, an α_2A-adrenoceptor antagonist; and ARC-239, an α_2B/2C⁻ and also α₁-adrenoceptor antagonist, blocked the increase of colonic temperature of the rabbit produced by 2 μg/kg LPS administered intravenously without being able in themselves to affect colonic temperature. In addition, prazosin, WB-4101 and CH-38083 antagonized the fall in skin temperature that occurred at the time when the colonic temperature was rising in control animals injected with LPS. All these results suggest that norepinephrine, through stimulation of both α₁⁻andα₂⁻ (α_2A⁻andα_2B/2C⁻) adrenoceptor subtypes, is involved in producing fever in response to bacterial LPS.     

Insulin stimulates membrane phospholipid metabolism by enhancing endogenousα1-adrenergic activity in the rat hippocampus

Insulin stimulates membrane phospholipid metabolism and activates protein kinase C (PKC) in its peripheral target tissues. Additionally, insulin can stimulate PKC activity in cultured fetal chick neurons. In the present study, we tested whether insulin can stimulate membrane phospholipid metabolism in the rat hippocampus, a CNS region in which insulin has been reported to stimulate the phosphorylation of a PKC substrate protein and to suppress spontaneous electrical activity of pyramidal cells. Concentrations of 1, 10 and 100 nM insulin significantly stimulated the accumulation of [³H]inositol phosphate ([³H]IP₁) and [³H]IP₂ in hippocampal slices labelled with [³H]myoinositol. Significant (P < 0.05) increases of hippocampal diacylglycerol (a product of phospoinositol hydrolysis) content were observed at 1, 5 or 10 min of incubation with 50 or 100 nM insulin. Addition of tetrodotoxin resulted in a suppression of insulin stimulation of [³H]IP₁ release, suggesting that insulin effects may be indirect and mediated via release of an endogenous neuronal transmitter within the hippocampus. Norepinephrine has been shown to both stimulate PI turnover and suppress the spontaneous electrical activity of pyramidal cells viaα₁-adrenergic receptors. Therefore, we tested whether the effects of insulin were mediated by norepinephrine. We measured [³H]IP₁ release in the presence or absence of theα₁-adrenergic antagonists prazobind and prazosin. These compounds blocked insulin stimulation of IP₁ accumulation, suggesting that the action of insulin to stimulate PI turnover is secondary to enhancement of endogenous noradrenergic activity within the hippocampus.