Properties of β-adrenoceptor sites in metabolizing and nonmetabolizing rat reticulocytes and in resealed reticulocyte ghosts

Summary Intact cells and resealed ghosts of a homogeneous reticulocyte population isolated from the blood of phenylhydrazine-treated rats bound -adrenergic ligands reversibly, stereospecifically and with high affinity. Maximal specific binding capacity under control conditions (37° C), corresponded to 9.9 ± 0.8 fmol/l cells ( 600 sites/cell) and was similar in intact cells and ghosts. Pretreatment with metabolic inhibitors decreased the density of binding sites in intact cells to 6.48 ± 1.1 fmol/l cells, but had no effect in ghosts. Incubation at 1°C reduced specific binding in paired experiments by 68 and 44% in intact cells and ghosts, respectively. Rewarming to 37°C increased specific binding in cells and ghosts by 270 and 190%, respectively. A temperature shift from 1 to 37°C reduced the K _D value for the antagonist (-)timolol from 8.6 ± 1.5 to 1.1 ± 0.3 nmol/l in intact cells, while no significant reduction in K _D was observed with ghosts. Under all conditions the receptor population was homogeneous with respect to antagonist affinity but inhomogenous with respect to agonist affinity. Low affinity agonist binding sites predominated in native cells and in GTP- loaded resealed ghosts (apparent K _D values 447 and 680 nmol/l, respectively). High affinity binding sites predominated in both preparations at 1°C (K _D 29 and 14 nmol/l, respectively). -Adrenoceptor sites in starved cells and ghosts at 37°C showed intermediate apparent K _D values. GTP had no effect on antagonist affinity or on the density of -adrenoceptors. The results suggest that intact metabolizing cells can regulate -adrenoceptor density by mechanisms which are not shared by ghost cells. The fractional contribution of high and low affinity states of the receptor to the overall binding of agonists seemed to be determined largely by the intracellular GTP concentration.     

Irreversible binding of a carbostyril-based agonist and antagonist to the β-adrenoceptor in DDT1 MF-2 cells and rat aorta

1. The chemoreactive ligands 5(2-(((1′-(4′-isothiocyanatophenylamino)thiocarbonyl)-amino)-2-methyl-propyl)amino-2-hydroxypropoxy)-3,4-dihydrocarbostyril (DCITC) and 8-hydroxy-5(2-(((1′-(4′-isothiocya-natophenylamino)thiocarbonyl)amino)-2-methylprop-2-yl)amino-1-hydroxyethyl)-carbostyril (HCITC)were synthesized and shown to be potent irreversible antagonist and agonist ligands, respectively, for the β-adrenoceptor in DDT₁ MF-2 (DDT) cells and the rat isolated aorta.
2. In DDT cell membranes DCITC and HCITC inhibited (−)[¹²⁵I]-iodocyanopindolol (CYP) binding to the β-adrenoceptor with IC₅₀ values of 1.1 and 18 nM, respectively. (−)-Isoprenaline inhibited [¹²⁵I]-CYP binding with an IC₅₀ of 355 nM. Pretreatment of membranes with either chemoreactive ligand produced a time- and concentration-dependent decrease in the β-adrenoceptor content, indicating irreversible receptor binding. DCITC at concentrations up to 10 μM did not stimulate cyclic AMP accumulation in DDT cells nor did it amplify forskolin-stimulated cyclic AMP accumulation.
3. In the rat isolated aorta, DCITC (0.1 μM) did not affect either the phenylephrine-mediated tissue contraction or the acetylcholine-mediated relaxation. DCITC attenuated the maximal (−)-isoprenaline-mediated relaxation of a phenylephrine contracted aorta in a concentration-dependent manner and shifted the dose-response curves for (−)-isoprenaline to the right. The DCITC-induced decrease in maximal response was not reversed by extensive tissue washing. By use of the operational model of agonism, the calculated dissociation constant for (−)-isoprenaline ws 286 nM and the estimated receptor reserve for this agonist was 23% at the maximal response.
4. HCITC and (−)-isoprenaline stimulated cyclic AMP accumulation in DDT cells with pD₂ values (negative logarithm to base 10 of EC₅₀) of 7.95 and 7.97, respectively, and both mediated the same maximal stimulation. In the rat isolated aorta, HCITC produced a concentration-dependent relaxation of the tissue with a pD₂ value of 6.62, whereas the pD₂ for (−)-isoprenaline was 7.03. However, HCITC produced a greater maximal relaxation of the tissue than (−)-isoprenaline. The HCITC-mediated stimulation of cyclic AMP accumulation and relaxation of the isolated tissue were blocked when the β-antagonist propranolol was added concurrently. In contrast, once the HCITC-mediated responses were established, the addition of propranolol did not result in any attenuation indicating that HCITC is an irreversible β-agonist.

     

μ-Opioid receptor and α2-adrenoceptor agonist binding sites in the postmortem brain of heroin addicts

The biochemical status of human brain -opioid receptors and ₂-adrenoceptors during opiate dependence was studied by means of the binding of [³H] [D-Ala², MePhe⁴, Gly-ol⁵] enkephalin (DAGO) and [³H]clonidine, respectively, in postmortem brains of heroin addicts who had died by opiate overdose or other causes. In the frontal cortex, thalamus and caudate of heroin addicts the density (B_max) and affinity (K_D) of -opioid receptors were similar to those in controls. In contrast, the density of ₂-adrenoceptors in heroin addicts was found to be significantly decreased in frontal cortex (B_max 31% lower), hypothalamus (B_max 40% lower) and caudate (B_max 32% lower) without changes in K_D values. When heroin addicts were divided into two subgroups according to the presence or absence of morphine in body fluids, only the group with positive screening for morphine showed relevant decreases in brain ₂-adrenoceptor density (B_max 36–48% lower), whereas the decreases in receptor density observed in the subgroup with negative screening for morphine did not reach statistical significance. The results suggest that desensitization of brain _2A-adrenceptors is a relevant adaptative receptor mechanism during opiate addiction in humans.     

Flerobuterol,aβ-adrenoceptor agonist,enhances serotonergic neurotransmission: an electrophysiological study in the rat brain

The two-adrenoceptor agonists salbutamol and clenbuterol have been shown to be effective antidepressant drugs. Flerobuterol, a new-adrenoceptor agonist, exhibits antidepressant activity in animal models. Given the long-standing notion that the serotonergic (5-HT) system might be involved in the etiology and/or the therapeutics of affective disorders and that this class of adrenergic agents can alter factors regulating 5-HT transmission, the effects of acute and repeated administrations of flerobuterol on the 5-HT system were studied. Acute administration of flerobuterol (up to 2 mg/kg, IV) did not modify the firing rate of dorsal raphe 5-HT neurons. However, the sustained administration of flerobuterol for two days (0.5 mg/kg/day, SC, delivered by an osmotic minipump) produced a marked decrease of the firing rate of 5-HT neurons. The reversal of this effect of flerobuterol by the somatodendritic 5-HT autoreceptor antagonist spiperone suggests that this decrease in the firing activity of 5-HT neurons in rats treated for 2 days with flerobuterol resulted from an enhanced synaptic availability of 5-HT. This initial decrease in firing activity of 5-HT neurons was followed by a progressive recovery to normal after 14 days of treatment with flerobuterol. At this point in time, the effect of intravenous lysergic acid diethylamide on the firing of 5-HT neurons was attenuated, indicating that the somatodendritic 5-HT autoreceptors had desensitized. The effectiveness of the electrical stimulation of the ascending 5-HT pathway in suppressing the firing activity of dorsal hippocampus pyramidal neurons was markedly enhanced in rats treated with flerobuterol for 14 days. This enhanced efficacy of the stimulation could not be ascribed to an increased sensitivity of the postsynaptic neurons to 5-HT, since their responsiveness to microiontophoretic applications of 5-HT was unaltered. Neither could this enhanced 5-HT signal transfer be attributed to a modification of the function of terminal 5-HT autoreceptors in controlling 5-HT release, since the decremental effect of increasing the frequency of stimulation (from 1–5 Hz) was similar in control and in flerobuterol-treated rats. In keeping with available data, it is most likely that the enhancement of 5-HT neurotransmission by long-term administration of flerobuterol is due to an increased availability of the neurotransmitter. It is concluded that the enhancement of 5-HT neurotransmission might underlie the therapeutic action of-adrenoceptor agonists in major depression.     

Differential region-specific regulation of central α1-adrenoceptor binding following chronic haloperidol and clozapine administration in the rat

Rationale Many antipsychotics exhibit potent anti-₁-adrenergic receptor activity, which has been suggested to contribute to typical and atypical antipsychotic effects and to the production of centrally mediated side effects.Objectives To assess the relative contribution of ₁-adrenoceptors to the mechanism of action of haloperidol and clozapine and to identify possible sites of action.Methods We examined the effect of chronic haloperidol and clozapine treatment on ₁-adrenoceptor characteristics in several rat brain regions. For comparison, D₂-like dopamine receptor density in the striatum was also determined.Results Clozapine administration (25 mg/kg/day i.p., 21 days) significantly increased ₁-adrenoceptor density in the frontal cortex (44%), remaining cortex (49%) and thalamus (93%) but binding levels in the hippocampus and spinal cord were unchanged relative to vehicle. Haloperidol treatment (1.5 mg/kg/day i.p., 21 days) also significantly increased the density of ₁-adrenoceptor binding in the thalamus (73%), but had no effect on ₁-adrenoceptor levels in any other region examined. ₁-Adrenoceptor affinity in the cortex was not significantly altered by either antipsychotic treatment. Haloperidol, in contrast to clozapine, significantly upregulated dopamine D₂-like binding in the striatum.Conclusions Central ₁-adrenoceptors are differentially regulated after chronic haloperidol and clozapine treatment. It is suggested that thalamic ₁-adrenoceptors may represent a common anatomical locus contributing to the antipsychotic activity and/or ₁-adrenoceptor centrally mediated side effects of both drugs, whereas the selective upregulation of cortical ₁-adrenoceptor density by clozapine may contribute, in part, to its superior atypical properties.     

The pharmacokinetics of theβ 2-adrenoceptor agonist fenoterol in healthy women

Summary We have studied the pharmacokinetics of fenoterol in healthy women during and after a 3 h intravenous infusion of different doses within the therapeutic range for tocolysis (0.5 g·min^–1, 1.0 g·min^–1, and 2.0 g·min^–1). A specific and sensitive radioimmuno-assay was used for the determination of fenoterol. For compartmental analysis the plasma concentration time data were fitted with the TOPFIT program, assuming two exponentials.The total clearance of fenoterol increased with dose (1299 ml·min^–1 at 0.5 g·min^–1, 1483 ml·min^–1 at 1.0 g·min^–1, and 1924 ml·min^–1 at 2.0 g·min^–1), as did the apparent volume of distribution (from 491 at the lowest to 851 at the highest dose).In contrast, the apparent half-lives were not dose-dependent, with t_{1/2· 1} 4.8 min and t_{1/2· 2} 52 min.This paper is dedicated to Prof. Dr. Ellen Weber, Heidelberg, FRG     

Molecular mechanisms for the persistent bronchodilatory effect of the β2-adrenoceptor agonist salmeterol

Background:

β₂-adrenoceptor agonists are effective bronchodilators. In vitro studies demonstrated long-lasting airway smooth muscle relaxation by salmeterol after washout, the quick disappearance of this effect in presence of antagonists and its recovery after antagonist removal. Current explanations invoke salmeterol accumulation in the membrane (‘diffusion microkinetic’ model) or the existence of salmeterol-binding ‘exosites’. An alternative model based on ‘rebinding’ of a dissociated ligand to the receptor molecules also produces an apparent decrease in the ligand''s dissociation rate in the absence of competing ligands.

Purpose and approach:

Computer-assisted simulations were performed to follow the receptor-occupation by a salmeterol-like ligand and a competing ligand as a function of time. The aptness of the models to describe the above in vitro findings was evaluated.

Key results:

The ‘diffusion microkinetic’ model is sufficient to explain a long-lasting β₂-adrenoceptor stimulation and reassertion as long as the membrane harbors a high concentration of the agonist. At lower concentration, ‘rebinding’ and, in second place, ‘exosite’ binding are likely to become operational.

Conclusions and implications:

The ‘rebinding’ and ‘exosite’ binding mechanisms take place at a sub-cellular/molecular scale. Pending their demonstration by experiments on appropriate, simple models such as intact cells or membranes thereof, these mechanisms remain hypothetical in the case of salmeterol. Airway smooth muscle contraction could also be governed by additional mechanisms that are particular to this macroscopic approach.     

Pharmacological profiles of a novel α1-adrenoceptor agonist,PNO-49 B,at α1-adrenoceptor subtypes

The effects of a newly synthesized compound, PNO-49B, (R)-(-)-3-(2-amino-l-hydroxyethyl)-4-fluo-romethanesulfonanilide hydrochloride, on ₁-adrenocepfor subtypes were examined in various tissues in which the following distribution of ₁-adrenoceptor subtypes has been suggested: dog carotid artery (_1B), dog mesenteric artery (_1N), rabbit thoracic aorta (_1B + _1L), rat liver (₁B), rat vas deferens (_1A + _1L), rat cerebral cortex (_1A + _1B) and rat thoracic aorta (controversial subtype).PNO-49 B (0.1–100 M) produced concentration-dependent contractions in dog mesenteric artery, rabbit thoracic aorta, rat thoracic aorta and rat vas deferens; and the maximal amplitudes of contraction were almost the same as or slighly less than those of noradrenaline. By contrast, the maximal response to PNO-49 B in dog carotid artery was markedly smaller than the response to noradrenaline. In rabbit thoracic aorta, the contractile response to PNO-49 B was not affected by inactivation of the _1B subtype with chloroethylclonidine (CEC), although the response to noradrenaline was attenuated by that treatment. The dissociation constants (K_A) of PNO-49 B were not different among the rat thoracic aorta, dog carotid and mesenteric arteries and rabbit thoracic aorta (CEC-pretreated). The contractile responses to PNO-49 B were inhibited competitively by prazosin, HV723 (-ethyl-3,4,5-trimethoxy--(3-((2-(2-methoxy-phenoxy)-ethyl)))-amino(propyl)benzeneacetonitrile fumarate) and by WB4101 (2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane). The estimated pA₂ values were high for prazosin and WB4101 in rat thoracic aorta and for HV 723 in dog mesenteric artery, whereas the pA₂ values for these three antagonists in rabbit thoracic aorta were low and were not altered by pretreatment with CEC. The binding of [³H]-prazosin to membranes prepared from rat vas deferens and liver was inhibited by PNO-49 B in a concentration-dependent manner. The resulting pK₁ value for the liver was approximately 1.5 log units lower (one thirtieth in affinity) than the values for the epididymal and prostatic portions of the vas deferens. PNO-49B also inhibited biphasically [³H]prazosin binding to prazosin-high affinity sites of rat cerebral cortex membranes, and the low but high affinity sites for PNO-49B was abolished by CEC-pretreatment. PNO-49B had no effect on the prejunctional ₂-adrenoceptors in rat vas deferens (prostatic portion) nor on the -adrenoceptors in rat atria. The contractile response to PNO-49 B in rat thoracic aorta was not inhibited by cimetidine, pyrilamine or ketanserin.These results indicate that PNO-49B is an ₁-adrenoceptor agonist with a lower affinity and/or efficacy at the _1B subtype as compared with other ₁-subtypes.     

On the assessment of the β-adrenoceptor blocking activity of propranololuUsing the rat isolated right ventricle

Two methods for determining pA₂ values, the Schild regression and the use of the formula pA₂ = pA_x + log(x − 1), have been reassessed. The effects of propranolol on the contractile responses of the rat isolated right ventricle to isoprenaline were studied. The inhibitory effect with the lower concentrations of propranolol tested (3 × 10⁻⁹–10⁻⁷ M) was solely competitive. Thus there was a parallel dextral displacement of the concentration-response curves to isoprenaline with no depression of the maximal responses. Dual antagonism was observed with the higher concentrations of propranolol tested (10⁻⁶ and 3 × 10⁻⁶ M), thereby showing a parallel displacement of the concentration-response curve to the lower concentrations of isoprenaline with a depression of the maximal responses. Without analysis of data for individual concentrations of propranolol on concentration-response curves to isoprenaline, Schild regression did not clearly illustrate that the antagonism was not solely competitive with the higher concentrations of propranolol. We suggest that data for individual concentrations of antagonist should always be analyzed to ascertain whether there has been a parallel shift of the concentration-response curves to agonist prior to determining pA₂ values.     

β-Adrenoceptor regulation and functional responses in the guinea-pig following chronic administration of the long-acting β2-adrenoceptor agonist formoterol

Formoterol is a long acting ₂-adrenoceptor agonist designed for the alleviation of the symptoms of asthma. This study examined the effects of 14 day administration of formoterol (200 g/kg/day i.p.) on ₁- and ₂-adrenoceptors in guinea-pig cardiac and lung tissue. Quantitative autoradiography was used to measure changes in receptor density and organ bath studies determined alterations in functional response.Formoterol treatment produced marked reductions of between 43% and 77% in ₂-adrenoceptor density in all regions of the heart (atrioventricular node, bundle of His, right and left bundle branches, interventricular and interatrial septa, right and left atria, ventricles and apex) and lung (bronchial and vascular smooth muscle and parenchyma) (P < 0.01, n = 6). ₁-Adrenoceptor density remained unchanged in all cardiac and lung regions. In functional studies (–)-isoprenaline was 4 fold less potent at causing relaxation of carbachol (1 M) precontracted tracheal smooth muscle (pD₂: control 8.49 ± 0.03, formoterol 7.91 ± 0.10, P < 0.001, n = 4), but formoterol treatment did not change the ability of (–)-isoprenaline to elicit a maximum response. The pK_B values for ICI 118,551, 7.33 ± 0.08 in the control and 7.20 ± 0.01 in formoterol treated animals, were between those expected for ₁- and ₂-adrenoceptors suggesting involvement of both subtypes in the response. In spontaneously beating right atria and electrically paced left atria, tissues in which responses are largely mediated by ₁-adrenoceptors, there was no significant change in responses to (–)isoprenaline (right atria pD₂: control 8.45 ± 0.02; formoterol 8.42 ± 0.11; P = 0.77, n = 4) (left atria pD₂: control 8.25 ± 0.03; formoterol 8.47 ± 0.08; P = 0.09, n = 4). In the presence of CGP 20712A (100 nM) the pK_B values did not change with formoterol treatment (left atria: control 9.59 ± 0.12, formoterol 9.66 ± 0.12; P = 0.70, n = 4) (right atria: control 8.93 ± 0.11, formoterol 9.11 ± 0.07; P = 0.25, n = 4).The doses and route of administration of formoterol used in this study differs from those used clinically. However, this study demonstrates that chrome formoterol administration produces selective down-regulation of ₂-adrenoceptors in the lung and heart. The changes in the lung are accompanied by a shift to the right in the concentration-response curve to -agonist stimulation with no change in the maximum response.     

Dorsal hand vein responses to the α₁-adrenoceptor agonist phenylephrine do not predict responses to the α₂-adrenoceptor agonist dexmedetomidine

Significant inter-individual variability exists in responses of human dorsal hand veins to activation of α-adrenoceptors. Simultaneous graded infusions of the α?- and α?-adrenoceptor agonists phenylephrine (3.66-8000 ng/min) and dexmedetomidine (0.0128-1000 ng/min) were given into dorsal veins of both hands and responses of 75 subjects were analyzed to assess whether a subject's sensitivity to phenylephrine (ED(50)) predicts his sensitivity to dexmedetomidine. Individual ED(50) estimates of dexmedetomidine and phenylephrine ranged between 0.06-412 and 14.2-7450 ng/min and exhibited only a weak positive relationship (r2 =0.074, P=0.018). Finger temperature, body mass index, age and phenylephrine sensitivity together accounted for about 30% of dexmedetomidine ED(50) variation (r2 =0.315, P<0.001). The large inter-individual variability observed in the responses of dorsal hand veins to both α?- and α?-adrenoceptor agonists is not explained by some common factors; instead, dorsal hand vein responsivity is separately determined for both receptor mechanisms.     

Pharmacological profile of the selective β3-adrenoceptor agonist mirabegron in cynomolgus monkeys

Mirabegron is a novel β₃-adrenoceptor agonist developed for the treatment of overactive bladder. To clarify the relationship between the pharmacological effects of mirabegron in monkeys and the clinical efficacy in patients with overactive bladder, the effect of mirabegron on bladder function was evaluated using cynomolgus monkeys. Quantitative PCR revealed that mRNA expression of β₃-adrenoceptors was most abundant (98 %) among β-adrenoceptor subtypes in the bladder of cynomolgus monkeys. Mirabegron, which showed selective and potent agonistic activity on monkey β₃-adrenoceptors expressed in Chinese hamster ovary cells with EC₅₀ value of 32 nmol/L and intrinsic activity of 0.8, induced concentration-dependent relaxation of bladder smooth muscle strips isolated from cynomolgus monkeys with EC₅₀ values of 120 nmol/L in 20 mmol/L KCl stimulation and 43 nmol/L under 9.81 mN resting tension. In conscious cynomolgus monkeys, mirabegron decreased micturition frequency at oral doses of 1 and 3 mg/kg and increased mean volume voided per micturition at an oral dose of 3 mg/kg. Plasma concentration at which bladder function improved in the cynomolgus monkeys was similar to that at the clinically effective dose in patients with overactive bladder. These data suggest that the relaxant function in monkey bladder is mainly mediated by β₃-adrenoceptors similar to that in the human bladder and mirabegron showed efficacy on the bladder functions of the same parameters in clinical evaluation endpoints.     

Interaction of β-adrenoceptor agonists with the serotonergic system in rat brain

Summary The effect of -adrenoceptor agonists on the behavioral effect of l-5-HTP in rats and mice was studied. All -agonists potentiated the behavioral syndrome elicited by l-5-HTP. However no indication for a correlation between their potencies to stimulate peripheral beta-receptors and their potencies to enhance l-5-HTP effects was found. The potentiating effect of salbutamol in rats was intensified by the MAO A inhibitor clorgyline, completely inhibited by (±)-propranolol and partly inhibited by WB-4101, while practolol was without effect. Lesions of 5-HT pathways by i.c.v. injections of 5,7-DHT impaired the potentiating effect of salbutamol in rats. In contrast, 6-OHDA lesions or alphamethyl-p-tyrosine pretreatment were without effect. A central site of action of salbutamol is suggested by the fact that it intensified l-5-HTP effects also after i.c.v. administration. Therefore the results suggest that salbutamol facilitates 5-HT transmission in rat brain probably via stimulation of central beta receptors.A part of this study was presented at the meeting of the Deutsche Pharmakologische Gesellschaft in Mainz, March 18–21, 1980     

A comparative study on the rat aorta and mesenteric arterial bed of the possible role of nitric oxide in the desensitization of the vasoconstrictor response to an α1-adrenoceptor agonist

1. In thoracic aortic strips with intact endothelium, the first and second (1 h later) dose-response curves obtained with methoxamine were almost the same.
2. Methoxamine caused a dose-dependent increase in perfusion pressure in the rat isolated mesenteric arterial bed, but the second (1 h later) dose-response curve for methoxamine showed a significant attenuation of the response in comparison with the first.
3. The attenuation shown by the second dose-response curve for methoxamine was significantly reduced, but not abolished, in mesenteric arterial beds without endothelium. Incubating endothelium-intact mesenteric arterial beds with N^G-nitro-L-arginine (L-NOARG) caused a significant, but not complete, reversal of the attenuation shown in the second dose-response curve.
4. Incubating the mesenteric arterial bed with capsaicin, tetrodotoxin, indomethacin or with isotonic high k⁺ (60 mM) plus nicardipine did not affect the above attenuation seen in the second dose-response curve.
5. The guanosine 3′:5′-cyclic monophosphate (cyclic GMP) level in the effluent from the perfused mesenteric arterial bed was significantly increased after the second exposure to methoxamine. This effect was significantly smaller after removal of the endothelium or pretreatment with L-NOARG.
6. These results suggest that a desensitization to methoxamine develops rapidly in the mesenteric arterial bed, but not in the aorta, and that release of nitric oxide from the endothelium plays a major role in this desensitization.

     

Effects of β-adrenoceptor antagonists on the firing rate of noradrenergic neurones in the locus coeruleus of the rat

Summary Acute i.v. administration of the non-selective -adrenoceptor antagonist dl-propranolol given in incremental doses (<40 mg/kg) did not affect the firing rate of locus coeruleus (LC) neurones in the rat, as revealed by single cell recording techniques. Furthermore, no effect was seen 4 h after a single i.p. dose of this -blocker (10 mg/kg). However, repeated treatment with dl-propranolol (1, 5, 10 or 20 mg/kg i.p., twice daily for 4 days) produced a significant, dose-dependent decrease of the average LC neuronal firing rate in comparison to controls. The dextro isomer of propranolol, which has negligible -blocking activity but the same local anaesthetic potency as the racemate, had no corresponding effect. The non-selective -adrenoceptor antagonist sotalol, which is one of the most hydrophilic -blockers, had much less inhibitory effect on LC neurones than dl-propranolol. The ₁-selective antagonist metoprolol did not change the firing of noradrenergic neurones in the LC after similar treatment for 4 days. However, when the rats were subjected to oral treatment for 28 days, metoprolol was found to produce a slight inhibitory effect although much less than dl-propranolol.In view of these findings we propose a stimulatory and mainly ₂-adrenoceptor-mediated control mechanism for the noradrencrgic neurones in the LC. This mechanism seems to be characterized by a delayed responsiveness.     

Brainβ-adrenoceptor binding sites in depressed suicide victims: effects of antidepressant treatment

-Adrenoceptor binding sites were measured by saturation binding of [³H]CGP 12177 in nine brain regions from 13 suicides, with a firm retrospective diagnosis of depression, who had been receiving antidepressant drugs, and 11 matched controls. Significantly lower numbers of-adrenoceptor binding sites were found in thalamus and temporal cortex (Brodmann area 38), but not in other brain regions, of antidepressant-treated suicides compared to controls. The lower number of-adrenoceptor binding sites in thalamus appeared to be related to drug treatment, whereas lower numbers of-adrenoceptors in temporal cortex were also found in antidepressant-free suicides.     

Aversive properties of theκ opioid agonist U50,488 in the week-old rat pup

Opioids have been shown to produce analgesia and to be reinforcing during the first week of life in the rat. Opioids also have analgesic actions in both the infant and adult, but can be aversive in the mature animal. We examined the aversive effects of the opioid agonist U50,488 during the first postnatal week in the rat pup in three ways. In the first experiment, U50,488, injected peripherally (1.0–30.0 mg/kg), was paired with an odor and pups were tested 8 h later for positional preference for avoidance of that odor. This task is similar to conditioned preference/aversion tests used with adult animals. Both 3- and 7-day-old pups learned to avoid the odor adulterated side at the two higher doses. When exposed to odors previously associated with U50,488, pups at both ages decreased locomotor activity. In a second experiment, acute treatment with U50,488 increased ultrasonic distress vocalizations (USV) equally at 3 and 7 days of age, increased locomotor activity, and decreased rectal temperature. Neither of the latter two effects was correlated with the increase in USV production. The third experiment showed that conditioned odor cues increased USV 8 h later in 3- and 7-day-old pups at 1.0–10.0 mg/kg without changes in activity or rectal temperature. The results from these studies suggest that U50,488 can produce aversions in the neonatal rat pup as it does in the adult.Supported by U.S.P.H.S. grant DA-06600     

Protective effects of the β3-adrenoceptor agonist CL316243 against N-methyl-D-aspartate-induced retinal neurotoxicity

We have previously reported that β₃-adrenoceptor agonists dilate retinal blood vessels, but their effects on retinal neurons have been unclear. In this study, we examined the action of the β₃-adrenoceptor agonist CL316243 against retinal damage induced by intravitreal injection of N-methyl-D-aspartate (NMDA) in rats. CL316243 was injected into the vitreous cavity before, with, or after intravitreal NMDA injection. Seven days after NMDA injection, cell loss in the ganglion cell layer (GCL) and thinning of the inner plexiform layer were observed. The reduction in the number of cells in the GCL was diminished by injection of CL316243 at 15, 30, 60, or 120?min after NMDA injection, whereas no significant protective effect was observed when CL316243 was administered 240?min after NMDA injection. Neither preinjection of CL316243 30?min before NMDA nor simultaneous injection of CL316243 with NMDA exerted any protective effect. The β₃-adrenoceptor antagonist L748337 almost completely abolished the protection conferred by CL316243 injection 120?min after NMDA injection. The number of parvalbumin-positive amacrine cells was decreased in eyes examined 1?day after NMDA treatment, but this was prevented by CL316243 injection at 120?min after NMDA injection. These results suggest that CL316243 exerts protective effects against NMDA-induced damage by stimulation of β₃-adrenoceptors. β₃-adrenoceptor agonists may be effective candidates for the treatment of retinal diseases associated with glutamate-induced excitotoxicity, including glaucoma and diabetic retinopathy.     

Effect of endothelium and carbachol on α-adrenoceptor agonist stimulated uptake and efflux of 45Ca in rat isolated aorta

Summary Preincubation with carbachol (10 M) did not affect basal ⁴⁵Ca accumulation by rat isolated aortic segments complete with endothelium, although ⁴⁵Ca accumulation was enhanced by removal of endothelium. This confirms the observation that in the presence of endothelium Ca²⁺ influx in rat aorta is antagonized, and indicates that the basal release of an endothelial derived factor might be sufficient to maximally antagonize basal Ca²⁺ influx, or alternatively that EDRF released as a result of muscarinic stimulation does not have identical effects to the factor released under basal conditions. Accumulation of ⁴⁵Ca stimulated by B-HT 920 but not that stimulated by phenylephrine was antagonized in the presence of endothelium. Contractions elicited by B-HT 920 were abolished in the presence of endothelium while contractions evoked by phenylephrine were reduced by about 50%. Preincubation with 10 M carbachol antagonized both phenylephrine (1 M) stimulated ⁴⁵Ca accumulation and contractile responses in the presence of endothelium to about the same extent. Therefore, it might be concluded that the inhibitory effect of EDRF in this tissue is due to an inhibition of stimulated Ca²⁺ influx. However, while addition of carbachol to tissues precontracted with phenylephrine elicited an immediate relaxation in the presence of endothelium, this relaxation could not be correlated with a reduction in tissue accumulation of ⁴⁵Ca. Carbachol also antagonized the phenylephrine-induced reduction of tissue ⁴⁵Ca content (i.e. efflux of Ca²⁺), in tissues preloaded with ⁴⁵Ca. This implies that the initial endothelial-mediated relaxant effect of carbachol in precontracted tissues cannot be explained either by reduced influx or by an enhanced efflux of Ca²⁺ from the smooth muscle cells. Therefore, it is concluded that although EDRF mediates a reduction in agonist-induced Cat²⁺ uptake it also exerts other effects, one of which may be an initial redistribution of intracelllar Ca²⁺, resulting in a reduction in free intracellular Ca²⁺ levels and therefore in relaxation. Send offprint requests to R. C. Miller     

Subeffective doses of nitroparacetamol (NCX-701) enhance the antinociceptive activity of the α₂-adrenoceptor agonist medetomidine

The α₂-adrenergic system is involved in pain processing and inflammation-induced sensitization. α₂-adrenoceptor agonists induce analgesia, and this effect is greater when administered in combination with other analgesics. In the present study, we assessed a possible enhancement of antinociception combining the α₂-adrenoceptor agonist medetomidine with subeffective doses of NCX701 (nitroparacetamol). The effects of the drugs were studied in spinal cord neuronal responses from adult male Wistar rats with carrageenan-induced inflammation, using the recording of single motor unit technique. The experiments showed that the i.v. administration of medetomidine and NCX701 induced a more potent and effective antinociceptive effect than medetomidine when given alone (ID₅₀: 0.47 ± 0.1 vs. 1.1 ± 0.1 μg/kg) or in the presence of paracetamol, in naturally-evoked nociceptive responses. In addition, the duration of antinociception was significantly longer (P < 0.001, 100 min after administration). The use of low doses of NCX701 and α₂-adrenoceptor agonists might open new perspectives in the treatment of inflammatory pain.