A role for presynaptic α2-adrenoceptors in angiotensin II-induced drinking in rats

Studies from this laboratory have shown that either central or peripheral administration of clonidine, the α₂-adrenoceptor agonist, can attenuate a variety of dipsogenic stimuli in rats. Further, yohimbine and tolazoline, α₂-adrenoceptor antagonists, augment the drinking response to both peripherally administered isoproterenol and angiotensin II. Studies reported here establish a dose-inhibition relationship between the dose of clonidine administered (2 to 32 μg/kg) intracerebroventricularly (IVT) and inhibition of the drinking response to peripherally administered angiotensin II (200 μg/kg, SC). DI₅₀ was approximately 4 μg/kg. Yohimbine (300 μg/kg, SC) reversed the antidipsogenic effect of centrally administered clonidine (32 μg/kg, IVT) on angiotensin II-induced (200 μg/kg, SC) water intake. Phenylephrine, an α₂-adrenoceptor agonist, administered IVT (40 and 80 μg/kg) also inhibited angiotensin II-induced drinking in a dose-related fashion. The antidipsogenic effect of phenylephrine (80 μg/kg) was blocked by administration of yohimbine (300 μg/kg, SC). Thus, this effect of phenylephrine most likely occurs by way of α₂-adrenoceptors. These results support a role for the pre-synaptic α₂-adrenoceptor in the mediation of drinking in rats. Activation of α₂-adrenoceptors is accompanied by reduced water intake while inhibition of these receptors enhances water intake.     

Microinjection of carbachol in the lateral hypothalamus produces opposing actions on nociception mediated by α1- and α2-adrenoceptors

Electrical stimulation of the lateral hypothalamus (LH) produces antinociception partially blocked by intrathecal α-adrenergic antagonists, but the mechanism underlying this effect is not clear. Evidence from immunological studies demonstrates that substance P-immunoreactive neurons in the LH project near the A7 catecholamine cell group, a group of noradrenergic neurons in the pons known to effect antinociception in the spinal cord dorsal horn. Such evidence suggests that LH neurons may activate A7 neurons to produce antinociception. To test this hypothesis, the cholinergic agonist carbachol was microinjected into the LH at doses of 63, 125 and 250 nmol and the resulting effects on tail-flick and nociceptive foot-withdrawal latencies were measured. All three doses significantly increased response latencies on both tests, with the 125-nmol dose providing the optimal effect. Intrathecal injection of the opioid antagonist naltrexone (97 nmol) partially reversed antinociception, but neither the α₂-adrenoceptor antagonist yohimbine nor the α₁-adrenoceptor antagonist WB4101 altered latencies. However, two sequential doses of yohimbine blocked LH-induced antinociception on both tests. In contrast, two sequential doses of WB4101 increased nociceptive responses on both the tail-flick and foot-withdrawal tests. These findings, and those of published reports, suggest that neurons in the LH activate spinally projecting methionine enkephalin neurons, as well as two populations of A7 noradrenergic neurons that exert a bidirectional effect on nociception. One of these populations increases nociception through the action of α₁-adrenoceptors and the other inhibits nociception through the action of α₂-adrenoceptors in the spinal cord dorsal horn.     

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.     

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.     

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.     

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.     

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.     

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.     

The role of α2-adrenoceptors of the medullary lateral reticular nucleus in spinal antinociception in rats

We attempted to find out the role of α₂-adrenoceptors of the medullary lateral reticular nucleus (LRN) in antinociception in rats. Spinal antinociception was evaluated using the tail-flick test, and supraspinal antinociception using the hotplate test. Antinociceptive effects were determined following local electric stimulation of the LRN, and following microinjections of medetomidine (an α₂-adrenoceptor agonist; 1–10 μg), atipamezole (an α₂-adrenoceptor antagonist; 20 μg) or lidocaine (4%) into the LRN. The experiments were performed using intact and spinalized Hannover-Wistar rats with a unilateral chronic guide cannula. Electric stimulation of the LRN as well as of the periaqueductal gray produced a significant spinal antinociceptive effect in intact rats. Medetomidine (1–10 μg), when microinjected into the LRN, produced no significant antinociceptive effect in the tail-flick test in intact rats. However, following spinalization, medetomidine in the LRN (10 μg) produced a significant atipamezole-reversible antinociceptive effect in the tail-flick test in the hot-plate test, medetomidine (10 μg) in the LRN produced a significant atipamezole-reversible increase of the paw-lick latency in intact rats. Microinjection of atipamezole (20 μg) or lidocaine alone into the LRN produced no significant effects in the tail-flick test. The results are in line with the previous evidence indicating brat the LRN and the adjacent ventrolateral medulla is involved in descending inhibition of spinal nocifensive responses. However, α₂-adrenoceptors in the LRN do not mediate spinal antinociception but, on the contrary, their activation counteracts antinociception at the spinal cord level. The spinal aninociceptive effect of supraspinally administered medetomidine in spinalized rats can be explained by a spread of the drug (e.g., via circulation) which then directly activates α₂-adrenoceptors at the spinal cord level.     

Idazoxan and brain α2-adrenoceptors in the rabbit

The effect of intravenous infusion of idazoxan on the depressor response to intracisternal clonidine 1 microgram/kg and on [3H]yohimbine binding in the fore- and hindbrain of the rabbit was examined. Idazoxan was infused either acutely (30 min) or chronically (5 days) at doses of 0.56 or 1.1 mg/h. Idazoxan 1.1 mg/h reduced the fall in blood pressure after clonidine. This attenuation of the depressor response was observed in the groups that were given the higher dose of idazoxan both acutely and chronically. The extent of attenuation was not modified by the duration of treatment. The low dose of idazoxan given acutely had no significant effect on the response to clonidine but the chronically infused group showed an enhanced response. A significant increase in the number of [3H]yohimbine binding sites (83%) was observed in the forebrain after 5 days infusion of 1.1 mg/h idazoxan with no change in the hindbrain. The lower dose of infusion did not cause any significant change in [3H]yohimbine binding in either brain region. Thus it appears that the susceptibility of the alpha 2-adrenoceptor binding sites to up-regulation by idazoxan may depend on the brain region observed.     

Subtype-specificity of the presynaptic α2-adrenoceptors modulating hippocampal norepinephrine release in rat

In vivo brain microdialysis and high-performance liquid chromatography with electrochemical detection were used to study the effect of different selective α₂-antagonists on hippocampal norepinephrine (NE) release in freely moving awake rat. Systemic administration (0.5 mg/kg i.p.) of either the α_2AD-antagonist BRL 44408 or the α-_2BC-antagonist ARC 239 did not significantly change the basal release of NE. At a higher dose (5 mg/kg i.p.) ARC 239 was still ineffective, whereas BRL 44408 caused a significant increase of the extracellular level of NE. Similar results were obtained from in vitro perfusion experiments. Rat hippocampal slices were loaded with [³H]NE and the electrical stimulation-evoked release of [³H]NE was determined. The α₂-antagonists were applied in a concentration range of 10⁻⁸ to 10⁻⁶ M. ARC 239 was ineffective, whereas BRL 44408 significantly increased the electrically induced release of [³H]NE. In agreement with the data of microdialysis and perfusion experiments. BRL 44408 displaced [³H]yohimbine from hippocampal and cortical membranes of rat brain with high affinity whereas ARC 239 was less effective. The pK_i values of eight different α₂-adrenergic compounds showed a very good correlation (r = 0.98, slope = 1.11 P < 0.0001) in hippocampus and frontal cortex where the α₂-adrenoceptors have been characterized as α_2D-subtype. Our data indicate that hippocampal NE release in rat is regulated by α_2D-adrenoceptors, a species variation of the human α_2A-subtype.     

Inhibition of spontaneous inhibitory postsynaptic currents (IPSC) by noradrenaline in rat supraoptic neurons through presynaptic α2-adrenoceptors

It has been shown that noradrenergic activation has great influence on the activities of hypothalamic supraoptic neurons. No direct evidence has been reported on the presynaptic effects of adrenoceptors in the actions of noradrenaline on supraoptic neurons, although postsynaptic mechanisms have been studied extensively. In the present study, we explored presynaptic effects of noradrenaline on the supraoptic neurons by measuring spontaneous inhibitory postsynaptic currents (IPSC) with the whole-cell patch-clamp technique. Noradrenaline reduced the frequency of IPSCs in a dose-dependent (10⁻⁹ to 10⁻³ M) and reversible manner. Noradrenaline did not affect the amplitude of IPSCs at concentrations of 10⁻⁹ to 10⁻⁵ M, but reduced the amplitude of IPSCs at high concentrations (10⁻⁴ and 10⁻³ M). The inhibitory effects of noradrenaline were mimicked by the α₂-agonist clonidine (10⁻⁴ M), but not by the α₁-agonist methoxamine (10⁻⁴ M) nor by the β-agonist isoproterenol (10⁻⁴ M). Moreover, the inhibitory effects of noradrenaline on IPSCs were blocked by the non-selective α antagonist phentolamine (10⁻⁴ M) or the selective α₂-antagonist yohimbine (10⁻⁴ M), but not by the α₁-antagonist prazosin (10⁻⁴ M). These results suggest that noradrenaline inhibits release of GABA from the presynaptic GABAergic terminals of the supraoptic neurons by activating presynaptic α₂-adrenoceptors and such presynaptic mechanisms may play a role in the excitatory control of SON neurons by noradrenergic neurons.     

Autoradiographic studies of central α2A- and α2C-adrenoceptors in the rat using [H]MK912 and subtype-selective drugs

In the present study we examined the distribution of α_2A- and α_2C-adrenoceptors in tissue slices from the rat cervical spinal cord and from brain slices collected at the level of the striatum. To differentiate between α_2A- and α_2C-adrenoceptors, the slices were incubated with [³H]MK912 in the presence of graded concentrations of the α_2A-selective drug, BRL44408, or the α_2C-selective drug, spiroxatrine. Computer analysis of the autoradiograms indicated that 0.4 nM [³H]MK912 plus 185 nM BRL44408 selectively labeled α_2C-adrenoceptors, while 0.4 nM [³H]MK912 plus 220 nM spiroxatrine selectively labeled α_2A-adrenoceptors. Using this approach, α_2C-adrenoceptors were detected in the striatum, while α_2A-adrenoceptors predominated in the cortical layers 1–4, the spinal cord distal dorsal horn, the septum and the endopiriform nucleus.     

α2 and α1-adrenoceptors mediate opposing actions on parasympathetic neurons

We used intracellular recording methods to analyze the membrane responses to norepinephrine in cat vesical parasympathetic ganglia. In parasympathetic neurons, norepinephrine (NE) produces a membrane hyperpolarization, a membrane depolarization often accompanied by cell firing and a biphasic potential, a hyperpolarization followed by a depolarization. We found that the NE hyperpolarization is mediated through alpha 2-adrenoceptors while the NE depolarization is mediated through alpha 1-adrenoceptors. This situation is different than in sympathetic neurons where beta-adrenoceptors mediate a NE depolarization.     

Chronic variable stress induces supersensitivity of central α2-adrenoceptors which modulate the jaw-opening reflex in the rat

In a previous study, we found that the sensitivity of central postsynaptic α₂-adrenoceptors which modulate, in an inhibitory way, the activity of the jaw-opening reflex (JOR) is reduced after chronic repeated stress (tail pinch) in the rat. The aim of this study was to assess the effects of exposure to a chronic variable stress regime on these adrenoceptors. To do this, the digastric electromyographic responses elicited by orofacial electrical stimulation after the intravenous administration of cumulative doses (×3.3) of the α₂-adrenoceptor agonist, clonidine (0.1–10 000 μg/kg), were recorded. As expected, in unmanipulated control rats, clonidine inhibited the reflex, in a dose-dependent manner, until abolition (ED₅₀=17.3±2.2 μg/kg). Single tail pinch did not significantly alter the ability of clonidine to abolish the reflex. However, chronic variable stress led to an enhancement of the inhibitory effect of clonidine on the amplitude of JOR, resulting in a shift to the left of the dose-response curve in comparison with that of the control group (ED₅₀ was reduced by 37%, P=0.032), without affecting either the estimated maximum effect for the agonist or the slope of the inhibitory function. This in vivo result indicates that chronic variable stress leads to an increased sensitivity of central α₂-adrenoceptors which modulate JOR, in contrast to the desensitization of these adrenoceptors found after repeated exposure to the same stressor.     

Substance P suppresses the activity of α2-adrenoceptors of the nucleus reticularis gigantocellularis involved in cardiovascular regulation in the rat

We evaluated possible interactions between substance P (SP) and the α₂-adrenoceptors in the nucleus reticularis gigantocellularis (NRGC) of the medulla oblongata involved in cardiovascular regulation. Adult, male Sprague-Dawley rats anesthetized with pentobarbital sodium (40 mg/kg, i.p., with 10 mg/kg/h i.v. supplements) were used. The circulatory suppressant efficacy of a centrally acting α₂-adrenoceptor agonist, guanabenz, was used as the experimental index. Bilateral microinjection of SP (300 or 600 pmol) into the NRGC, a medullary site that is critically involved in the cardiovascular depressive actions of guanabenz, significantly diminished the hypotensive and bradycardiac efficacy of the aminoguanidine compound (100 μg/kg, i.v.). This implied reduction in α₂-adrenoceptor activity in the NRGC by SP was antagonized by its selective receptor antagonist, [d-Pro²,d-Trp^7,9]-SP (1200 pmol). Similarly, attenuation by SP of the cardiovascular suppressant effects of guanabenz was also reversed by immunocytochemically verified depletion of dopamine-β-hydroxylase-immunoreactive nerve terminals in the NRGC, elicited by the selective noradrenergic neurotoxin, DSP4 (50 μg). These data suggest that SP may exert an inhibitory action on the α₂-adrenoceptors in the NRGC that are involved in central cardiovascular regulation, possibly via a presynaptic modulation on noradrenergic neurotransmission.     

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.     

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.     

β2-glycoprotein I, anti-β2-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.     

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.