β2-glycoprotein I: antiphospholipid syndrome and T-cell reactivity

There is increasing evidence showing that recurrent thrombosis and intrauterine fetal loss in antiphospholipid syndrome (APS) are attributable to antiphospholipid (aPL) antibodies. We have recently identified autoreactive CD4⁺ T cells to β₂-glycoprotein I (β₂GPI) that promote production of pathogenic antiphospholipid antibodies. β₂GPI-specific CD4⁺ T cells preferentially recognize the antigenic peptide containing the major phospholipid (PL)-binding site in the context of DR53. T-cell helper activity that stimulates B cells to produce IgG anti-β₂GPI antibodies is mediated through IL-6 and CD40–CD154 interaction. β₂GPI-specific T cells respond to reduced β₂GPI and recombinant β₂GPI fragments produced in a bacterial expression system but not to native β₂GPI, indicating that the epitopes recognized by β₂GPI-specific T cells are ‘cryptic’ determinants, which are generated at a subthreshold level by the processing of native β₂GPI under normal circumstances. Although β₂GPI-specific T cells are detected in both APS patients and healthy individuals, these autoreactive T cells are activated in vivo in APS patients but not in healthy individuals. These findings indicate activation of β₂GPI-specific T cells and subsequent production of pathogenic anti-β₂GPI antibodies can be induced by the exposure of such T cells to cryptic peptides of β₂GPI efficiently presented by functional antigen-presenting cells (APC). Delineating the mechanisms that induce the efficient processing and presentation of cryptic determinants of β₂GPI as a consequence of antigen processing would clarify the etiology that initiates the autoantibody response in APS.     

Anti-β2-glycoprotein I antibodies—When and how should they be measured?

The biological criteria of the antiphospholipid syndrome defined at the Sapporo meeting in 1998 included the presence of lupus anticoagulant (LA) and/or anticardiolipin antibodies at medium and high titers. During the 48th SSC meeting held in Boston July 2002, it was proposed to modify these criteria. Four patient groups were defined, the first one comprising LA and anti-β₂glycoprotein I antibodies (aβ₂GPI), the second one LA only, the third one aβ₂GPI only and the fourth one other antiphospholipid antibodies such as antiprothrombin, anticardiolipin, antiphosphatidylethanolamine, etc. This proposition raised the issue of the association of aβ₂GPI with APS clinical criteria (thrombosis and pregnancy morbidity). In some studies, a strong association between IgG aβ₂GPI and thrombosis was found, whereas in others this association could not be demonstrated. In the obstetrical field, few studies are available and no clear conclusion can be drawn yet. However, for thrombosis or pregnancy morbidity, it has been shown that in up to 10% of patients, aβ₂GPI are the sole antibodies present and therefore the diagnosis of APS would be missed in these patients. In addition, some studies suggest that the severity of disease is dependent on the number of positive tests and on their titers. We recommend aβ₂GPI assays to be included in the panel of antiphospholipid screening tests. However, the standardisation of aβ₂GPI assays has to be improved in order to ensure better comparability between the studies.     

Beta2-glycoprotein I, anti-beta2-glycoprotein I, and fibrinolysis

β₂-glycoprotein-I (β₂GPI) is a phospholipid-binding plasma protein that consists of five homologous domains. Domain V is distinguished from others by bearing a positively charged lysine cluster and hydrophobic extra C-terminal loop. β₂GPI has been known as a natural anticoagulant regulator. β₂GPI exerts anticoagulant activity by inhibition of phospholipid-dependent coagulation reactions such as prothrombinase, tenase, and factor XII activation. It also binds factor XI and inhibits its activation. On the other hand, β₂GPI inhibits anticoagulant activity of activated protein C. According to the data from knockout mice, β₂GPI may contribute to thrombin generation in vivo. Phospholipid-bound β₂GPI is one of the major target antigens for antiphospholipid antibodies present in patients with antiphospholipid syndrome (APS). Binding of pathogenic anti-β₂GPI antibodies increases the affinity of β₂GPI to the cell surface and disrupts the coagulation/fibrinolysis balance on the cell surface. These pathogenic antibodies activate endothelial cells via signal transduction events in the presence of β₂GPI. Impaired fibrinolysis has been reported in patients with APS. Using a newly developed chromogenic assay, we demonstrated lower activity of intrinsic fibrinolysis in euglobulin fractions from APS patients. Addition of monoclonal anti-β₂GPI antibodies with β₂GPI also decreased fibrinolytic activity in this assay system. β₂GPI is proteolytically cleaved by plasmin in domain V (nicked β₂GPI) and becomes unable to bind to phospholipids, reducing antigenicity against antiphospholipid antibodies. This cleavage occurs in patients with increased fibrinolysis turnover. Nicked β₂GPI binds to plasminogen and suppresses plasmin generation in the presence of fibrin, plasminogen, and tissue plasminogen activator (tPA). Thus, nicked β₂GPI plays a role in the extrinsic fibrinolysis via a negative feedback pathway loop.     

α2-Macroglobulin is an astroglia-derived neurite-promoting factor for cultured neurons from rat central nervous system

The neurite promoting factors in the astroglial conditioned medium (As-CM) were characterized by using primary cultures of embryonic rat neocortical neurons. The factors in the As-CM bind to lectins such as wheat germ agglutinin (WGA), suggesting that they contain sugar moieties. When the WGA-bound fractions were applied on a Superose 6 column, the activity was recovered mainly in two fractions, peak I and peak II. The peak II fraction was further purified by Mono Q anion exchange chromatography. A single protein band of 180 kDa was detected in the final Mono Q fraction by sodium dodesylsulfate polyacrylamide gel electrophoresis. The molecular weight coincided with that ofα₂-macroglobulin (α₂M). Western blotting showed that the single protein band was reacted with anti-α₂M antibody but not with anti-fibronectin and anti-laminin antisera. The neurite-promoting activity of the Mono Q fraction was inhibited by anti-α₂M antibody. Furthermore, commercially available α₂M also promotes neurite outgrowth in our assay system. These results strongly suggested that α₂M is one of the neurite-promoting factors in the As-CM.     

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.     

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.     

Immunoelectron microscopy of α2-glycoprotein: An astrocyte-specific antigen

The cellular and fine structural localization of the soluble brain-specific acidic α₂-glycoprotein was investigated using the indirect immunohistochemical method. The electron microscope was used to unambiguously identify cells containing the antigen.A single type of cell, the astrocyte, was found to be labelled with specific antisera directed against α₂-glycoprotein. Immunoperoxidase reaction product was found in astrocyte perikarya, their processes and perivascular end feet. It was found to be apparently associated with the cytoplasmic surface of mitochondria, reticular membranes and the plasma membrane. No specific labelling of neurones, oligodendrocytes, myelin or capillary endothelial cells was observed. The data is discussed in relation to the immunological properties of α₂-glycoprotein already reported.     

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.     

Elevated plasmin-α2-antiplasmin complex levels in hereditary angioedema: Evidence for the efficiency of the intrinsic fibrinolytic system

The contact activation and fibrinolytic systems were assessed in 5 patients with hereditary angioedema (HAE). Reductions in F XII levels and increase in kallikrein-like activity in some patients indicated activation of the contact (intrinsic) system of coagulation. A great increase in plasmin-α₂-antiplasmin complex in all subjects indicated that in this disease, there is a constantly ongoing fibrinolysis.Since C1-inhibitor, the deficient protein in HAE, is a poor inhibitor of the well-known extrinsic (tissue-type) plasminogen activator, but the major inhibitor of the contact activation system and a related phenomenon termed intrinsic fibrinolysis, our data show that this fibrinolytic system is also sometimes operating efficiently . Furthermore, the known clinical data on HAE are compatible with a role of intrinsic fibrinolysis in the pathophysiology of this disease.     

Noradrenaline- and time-dependent changes in neocortical α2- and β1-adrenoceptor binding in dorsal noradrenergic bundle-lesioned rats

A 6-hydroxydopamine-induced lesion of the dorsal noradrenergic bundle (DNB) in rats markedly decreased neocortical noradrenaline concentration (NNC) by 72–100% as measured 1, 3 and 13 months after the lesioning procedure. The concomitant assessment of neocortical α₂- and β₁-adrenoceptor binding (NAAB and NBAB, respectively) usually indicated significant increases of 25–74% for these two variables. There were, however, cases of unchanged NAAB and NBAB which presumably reflected an incomplete DNB lesion and a consequent time-related, partial recovery of NNC. The results emphasize the potential for long-term sequelae of the DNB lesion, and the existence of a critical NNC threshold (10–30% of control NNC values) which modulates postsynaptic α₂ and β₁-adrenoceptor density.     

Spatial working memory improvement by an α2-adrenoceptor agonist dexmedetomidine is not mediated through α2C-adrenoceptor

1. Aged α_2C-adrenoceptor knockout and wild type mice were used to investigate whether α_2C-adrenoceptors are involved in mediating the beneficial effects of α₂-adrenoceptor agonist, dexmedetomidine, on spatial working memory.

2. A win-stay task in the radial arm maze was used to dissociate the effects of dexmedetomidine on working vs. reference memory. In addition, the animals were tested in simple response habit learning in the T-maze.

3. Knockout mice made more working memory errors after the change of the baited arm in radial arm maze, but after training reached again as accurate level of performance as wild type controls. Dexmedetomidine 5 and 10 μg/kg alleviated the increase in spatial working memory errors after the change of the baited arm in knockout mice. Knockout and wild type mice performed equally well in T-maze, and dexmedetomidine had no effect on this simple response learning.

4. The present results indicate that α₂-adrenoceptor agonists have a selective effect on spatial working memory not only in monkeys but also in mice. Further, this study confirms our earlier finding that the presence of α_2C-adrenoceptors is not necessary for the spatial working memory enhancing effect of α₂-adrenoceptor agonists.

Distribution of Lys-γ2-melanocyte-stimulating hormone-(Lys-γ2-MSH)-like immunoreactivity in neuronal elements in the brain and peripheral tissues of the rat

Using an antiserum raised against Lys-γ₂-melanocyte-stimulating hormone (Lys-γ₂-MSH), with a high specificity for this peptide and its des-Lys derivative, γ₂-MSH, we found Lys-γ₂-MSH-like immunoreactivity to have a widespread distribution in the rat brain. In colchicine-treated rats, groups of immunopositive cell bodies were found in the intermediate and anterior lobes of the pituitary gland, in the hypothalamic arcuate nucleus and in the commissural part of the nucleus of the solitary tract (NTS). Immunopositive fibers were found to originate from the latter two cell body regions. The distribution of these fibers was similar to that of the pro-opiomelanocortin-containing cell bodies and projections as it has been described previously. Immunopositive terminals were found in brain regions containing neurons which have been shown to express mRNA for melanocortin receptors, though the distribution of Lys-γ₂-MSH-like immunoreactivity is considerably more widespread than that of mRNA for the ‘γ-MSH receptor’ (the melanocortin MC₃ receptor), which has been reported to be mainly expressed in the hypothalamus. In the periphery Lys-γ₂-MSH immunoreactivity was localized in the adrenal medulla and in neuronal fibers and varicosities in the heart. The vascular system, the bronchi and kidney were immunonegative. The occurrence of Lys-γ₂-MSH immunoreactivity in many of the brain regions which are involved in cardiovascular regulation offers leads for further studies on the putative role of γ-MSHs in cardiovascular control. The occurrence in the rat heart of Lys-γ₂-MSH-containing fibers suggests a role of the γ-MSHs in cardiac function.     

The subunits of α2-macroglobulin receptor/low density lipoprotein receptor-related protein, native and transformed α2-macroglobulin and interleukin 6 in Alzheimer's disease

To explore the role of α₂-macroglobulin receptor/low density lipoprotein receptor-related protein (α₂M-R/LRP) and its ligands in the pathogenesis of Alzheimer's disease (AD), antibodies were raised against its α- and β-subunits and their expression pattern in the CNS in AD and control cases was correlated with that of native and transformed α₂-macroglobulin (α₂M) and interleukin 6 (IL-6). The transmembranous β-subunit of α₂M-R/LRP and transformed α₂M were found in plaque cores in AD. Extramembranous α-subunit and native α₂M immunoreactivities were localized in activated plaque-associated astrocytes and extracellulary in plaques. IL-6 immunostaining was associated with neurofibrillary changes, and was also found extracellularly in the center of plaques and in microglial cells. Our finding that plaque cores contain a second transmembranous protein fragment, the β-subunit of α₂M-R/LRP, suggests ongoing membrane-protein degradation. By altering clearance and scavenger-like functions, fragmentation and breakdown of α₂M-R/LRP may have an important role in extracellular amyloid deposition and the formation of neurofibrillary tangles in AD.     

Activation of supraoptic magnocellular neurons by gamma2-melanocyte stimulating hormone (γ2-MSH)

The effects of intracarotid infusions of the peptide gamma₂-melanocyte stimulating hormone (γ₂-MSH) on electrophysiologically and immunohistochemically identified supraoptic nucleus (SON) units were investigated Over a wide dose range this agent always excited SON units, while control infusions of vehicle had no effect. Because neural responses invariably preceded blood pressure elevation, it appears that γ₂-MSH excitation of the magnocellular system was due to a direct effect on the central nervous system and was not a result of systemic cardiovascular responses. These results suggest a forebrain γ₂-MSH sensitive site in the activation of SON magnocellular neurons.     

Modulation of rat brainα2- and β-adrenergic receptor sensitivity following long-term treatment with antidepressants

After 7 days of treatment with a variety of antidepressant drugs (desipramine, imipramine, clomipramine, nortriptyline, nialamide), both an increase inα₂-receptor density and a decrease in β-receptor density were observed in the cerebral cortex but not limbic forebrain. However, mianserin caused a marked increase inα₂-receptors without any change in β-receptors. Nisoxetine did not produce any change in these two adrenergic receptors. It is suggested that intrasynaptic norepinephrine is important but that, in addition, other factors may be involved in the increase inα₂-receptors induced by antidepressant drugs.     

Specific bindings of prostaglandin D2, E2 and F2α in postmortem human brain

Binding activities specific for each of [³H]prostaglandin (PG) D₂, E₂ and F_2α were detected in the P₂ fraction of the human brain homogenates. The bindings were time-dependent, saturable and of high affinity;K_dvalues were 30 nM for all the PG bindings. Regional distribution of these binding activities was determined by measuring specific bindings with 10 nM [³H]PG-D₂, [³H]PG-E₂ and [³H]PG-F_2α in the P₂ fractions from 17 brain regions. The PG-D₂ binding activity was high in the hypothalamus, amygdala and hippocampus followed by cerebellar nuclei, thalamus, nucleus accumbens and cerebral cortex. The PG-E₂ binding sites were similarly concentrated in the hypothalamus and the limbic system, but, unlike the PG-D₂ binding, no significant binding of [³H]PG-3₂ was observed in cerebellar nuclei, cerebellar cortex and putamen. Compared with these two PG bindings, PG-F_2α binding activity was low in many areas, but significant binding was detected in the amygdala, cingulate cortex, cerebellar medulla, hippocampus, nucleus accumbens, midbrain and hypothalamus. These results suggest the presence and specific distribution of three distinct types of PG binding activities, i.e. specific binding of PG-D₂, PG-E₂ and PG-F_2α, in the human brain.     

Quantitative light microscopic autoradiographic localization of α2-adrenoceptors in the human brain

In the present work the anatomical distribution of α₂-adrenoceptors in the human central nervous system was studied in detail by quantitative autoradiography using the selective α₂ agonist [³H]bromoxidine ([³H]UK-14304) as a ligand. Only postmortem tissues from subjects free of neurological disorders were used in this study. Very high or high densities of α₂-adrenoceptors were found along layers I and III in non-visual neocortex, layers III and IVc of the visual cortex, CA₁ field — stratum lacunosum-moleculare — and dentate gyrus — stratum granularis — at the hippocampal formation, nucleus arcuatus at the hypothalamus, locus ceruleus, nucleus dorsalis of vagus and at the stratum granularis of the cerebellar cortex. Relevant densities of α₂-adrenoceptors were also observed along the remaining layers of neocortex, nuclei centralis, medialis and corticalis at the amygdala, anterior thalamic group and rotundocellularis nuclei, paraventricular and ventromedial hypothalamic nuclei, substantia innominata, superior colliculus — stratum zonale — and lateral periaqueductal area at the midbrain, nucleus tractus solitarii and dorsal horn — substantia gelatinosa — of the spinal cord. [³H]Bromoxidine specific binding was very low or negligible in the remaining brain areas. Although a general parallelism between the distribution of these receptors could be observed for the rat and human brain, dramatic species differences in the level of α₂-receptors were found in several brain areas, such as thalamus, amygdala or cerebellar cortex. In general, the distribution of α₂-adrenoceptors in the human brain found here was parallel to that described for the noradrenergic presynaptic terminals in the mammalian central nervous system, lending some weight to the proposed predominant presynaptic localization of these receptors. The relevance of the anatomical distribution of α₂-adrenoceptors in the human brain for a better knowledge of the neurochemistry of neuropsychiatric disorders is discussed.     

Relationships between β- and α2-adrenoceptors and G coupling proteins in the human brain: effects of age and suicide

Interactions between brain α₂- and β-adrenoceptors are of interest in physiological (aging) and pathological (major depression) processes involving both receptors. In this study, total β-adrenoceptors and β_1/2-subtypes were quantitated in postmortem human brains to investigate their relationships with α_2A-adrenoceptors and specific G proteins during the process of aging and in brains of suicide victims. Analysis of [³H]CGP12177 binding, in the presence of CGP20712A (β₁-antagonist), indicated that the predominant β-adrenoceptor in the frontal cortex is the β₁-subtype (65–75%). The density of total β- (r=−0.60, n=44) or β₁-adrenoceptors (r=−0.78, n=22), but not the β₂-subtype, declined with aging (3–80 years). The density of total β- or β₁-adrenoceptors, but not the β₂-subtype, correlated with the number of α₂-adrenoceptors quantitated in the same brains with the agonist [³H]UK14304 (r=0.71–0.81) or the antagonist [³H]RX821002 (r=0.61–0.66). Interestingly, the ratios α₂/β- or α₂/β₁-adrenoceptors did not correlate with the age of the subject at death, indicating that the proportion of α₂/β-adrenoceptors in brain remains rather constant during the process of aging. The density of β-adrenoceptors correlated with the immunodensity of Gαs (r=0.55) and Gβ (r=0.61) proteins, and that of α₂-adrenoceptors with those of Gαi_1/2 (r=0.88) and Gβ (r=0.65). In brains of suicides, compared to controls, the ratio between α₂- and β- or β₁-adrenoceptors (α₂-full agonist sites/β-sites) was greater (1.3- to 2.0-fold; P<0.05). The results demonstrate a close interdependence between brain α₂- and β-adrenoceptors during aging, and in brains of suicides. The quantitation of the α_2A/β-adrenoceptor ratio could represent a relevant neurochemical index in the study of brain pathologies in which both receptors are involved.     

Influence of centrally administered α- and γ2-melanocyte-stimulating hormone on hypothalamic blood flow autoregulation in the rat

The effect of intracerebroventricularly (i.c.v.) administered α-melanocyte-stimulating hormone (MSH) and γ₂-MSH on hypothalamic blood flow autoregulation was studied in anesthetized rats at different levels of standardized arterial hypotension. Autoregulation was impaired upon i.c.v. administration of 5 γ g/kg γ₂-MSH while α-MSH caused no change.. Since this effect of γ₂-MSH wa identical to that produced by i.c.v. naloxone in the same model, γ₂-MSH may be a functional antagonist of central opioid mechanisms participating in the control of cerebral blood flow autoregulation.     

Effect of α2 adrenergic agents upon central etorphine antinociception in the cat

Systemic (s.c.) administration of α₂ agonists clonidine (25–100 μg/kg) or guanfacine (50–400 μg/kg) elicited antinociception as assessed by the cat tail-flick model and potentiated in a dose-dependent manner the antinociceptive effect of etorphine (2.5 μg) administered directly into the periaqueductal gray. Conversely, systemic yohimbine (1 mg/kg) attenuated the effects of central etorphine, and diminished potentiation of etorphine by theα₂ agonists. Prior microinjection of clonidine (5μg) or guanfacine (5 μg) into the locus coeruleus (LC) reduced the intensity of central etorphine antinociception whereas central yohimbine (20 μg) pretreatment increased peak antinociceptive activity and prolonged the duration of etorphine. Thus, systemicα₂ agonists are inherently antinociceptive and potentiate central narcotic antinociception; however, the site of interaction betweenα₂ agonists and opiates does not appear to be the LC inasmuch asα₂ agonists attenuate the antinociceptive effect of etorphine when administered directly into the LC. A spinal site of action is suggested based upon known LC-spinal projections and our experimental observations.