Modulation of rat brain α-adrenoreceptor populations four weeks after stimulation of the nucleus locus coeruleus

We previously showed that electrical stimulation of the nucleus locus coeruleus was followed 4 weeks later by a greatly improved performance in the acquisition of a food-reinforced operant task. To ascertain whether adrenergic receptors were involved in this long-term behavioral modification, we studied the characteristics of the ₁, ₂, and -adrenoreceptors of the cerebral cortex 4 weeks after stimulation of the locus coeruleus. This stimulation induced a slight (14%) but significant increase in the number of ₁-receptor [(³H) WB 4101 binding sites] as well a rise in the number of ₂-receptor [(³H) clonidine binding sites]. The later rise mainly affected high-affinity ₂ sites (36%) and the number of low-affinity sites remained unchanged. No significant alteration in the number of -receptors [(³H)-dihydroalprenolol binding sites] was observed. To confirm this biochemical result, the effect of very small doses of clonidine (1, 2.5, 5 and 10 g/kg) was tested on locomotor activity in the open-field. In rats stimulated 4 weeks before injection, clonidine induced a biphasic effect, comprising firstly sedation which occurred 30 min after injection, and secondly, long-term hyperactivity which began 24 h after injection. For the 5 g/kg dose, this rebound of activity was detectable 8 days after injection. In implanted, control rats, only the sedative effect was observed. These findings are interpreted in relation to the current theories about -adrenoreceptors.     

Enhancement of brain kynurenic acid production by anticonvulsants--novel mechanism of antiepileptic activity?

In this study, we describe the effect of antiepileptic drugs on the production of kynurenic acid in rat cortical slices, and on the activity of kynurenic acid biosynthetic enzymes, kynurenine aminotransferases (KATs I and II) in the brain tissue. Phenobarbital, felbamate, phenytoin and lamotrigine (all at 0.5-3.0 mM) enhanced kynurenic acid production in vitro, and stimulated the activity of KAT I. In contrast, vigabatrin, gabapentin and tiagabine inhibited kynurenic acid synthesis in cortical slices with IC(50) of 3.9 (2.8-7.9), 3.7 (2.5-5.4) and 7.5 (3.5-14.3) mM, respectively. Vigabatrin, gabapentin and tiagabine reduced also the activity of KAT I with IC(50) of 1.6 (1.1-2.4), 0.1 (0.01-0.15), 0.9 (0.7-1.2) mM, and the activity of KAT II with IC(50) values of 6.0 (4.8-7.5), 0.2 (0.1-0.3) and 2.0 (1.5-2.6) mM, respectively. In conclusion, the enhancement of kynurenic acid formation displayed by carbamazepine, phenytoin, phenobarbital, felbamate and lamotrigine seems to be a novel mechanism, synergistic with other actions of these drugs, and potentially valuable in terms of better control of epilepsy.     

Activation and desensitization by cyclic antidepressant drugs of α2-autoreceptors, α2-heteroreceptors and 5-HT1A-autoreceptors regulating monoamine synthesis in the rat brain in vivo

The effects of antidepressant drugs on the synthesis of noradrenaline and serotonin (5-HT) were assessed using the accumulation of 3,4-dihydroxyphenylalanine (dopa) and 5-hydroxytryptophan (5-HTP) after decarboxylase inhibition as a measure of the rate of tyrosine and tryptophan hydroxylation in the rat brain in vivo. Three inhibitory synthesis-modulating receptors were investigated simultaneously: the alpha2C-autoreceptor modulating dopa/noradrenaline synthesis, and the alpha2A-heteroreceptor and 5-HT1A-autoreceptor modulating 5-HTP/5-HT synthesis. Acute treatment (2 h, i.p.) with desipramine (1-10 mg/kg), protriptyline (0.3-10 mg/kg) and nisoxetine (3-10 mg/kg), selective NA reuptake blockers, dose-dependently decreased dopa synthesis in cortex (15%-40%) and hippocampus (20%-53%). Fluoxetine (1-10 mg/kg) and zimelidine (1-10 mg/kg), selective 5-HT reuptake blockers, did not alter dopa synthesis. Fluoxetine and zimelidine dose-dependently decreased 5-HTP synthesis in cortex (14%-43%) and hippocampus (27%-54%). Desipramine and protryptyline did not alter 5-HTP synthesis in cortex but in hippocampus it was decreased (36%). Repeated desipramine (10 mg/kg for 1-21 days) or fluoxetine (3 mg/kg for 3-21 days) treatment resulted in a time-dependent loss in their ability to decrease dopa or 5-HTP synthesis. Desipramine (1-21 days) did not alter 5-HTP synthesis in cortex, but in hippocampus it was decreased (21%-37%, days 1-14) followed by recovery to control values (day 21). Fluoxetine (3-21 days) did not alter brain dopa synthesis. To further assess the desensitization of alpha2C-autoreceptors, alpha2A-heteroreceptors and 5-HT1A autoreceptors regulating the synthesis of dopa/NA or 5-HTP/5-HT after chronic desipramine and fluoxetine, the effects of clonidine (agonist at alpha2-auto/heteroreceptors) and 8-OH-DPAT (agonist at 5-HT1A-autoreceptors) were tested. In saline-treated rats, clonidine (1 mg/kg, 1 h) decreased dopa and 5-HTP synthesis in cortex (58% and 54%) and hippocampus (54% and 42%). In desipramine-treated rats (10 mg/kg, 21 days), but not in fluoxetine-treated ones (3 mg/kg, 14 days), the effect of clonidine was attenuated in cortex (12% and 18%) and only for dopa synthesis in hippocampus (31%). In saline-treated rats, 8-OH-DPAT (1 mg/kg, 1 h) decreased 5-HTP synthesis in cortex (63%) and hippocampus (75%). In fluoxetine-treated rats, but not in desipramine-treated ones, this inhibitory effect was markedly attenuated in cortex (26%) and hippocampus (9%). These findings indicate that acute treatment with cyclic antidepressant drugs results in activation of inhibitory alpha2C-autoreceptors, alpha2A-heteroreceptors and/or 5-HT1A-autoreceptors regulating the synthesis of dopa/NA and/or 5-HTP/5-HT in brain, whereas chronic treatment with these drugs is followed by desensitization of these presynaptic receptors.     

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     

Affinities of full agonists for cardiac β-adrenoceptors calculated by use of in vitro desensitization

Summary Positive chronotropic and inotropic responses of guinea-pig isolated spontaneously beating right atria and paced left atria to -adrenoceptor agonists were recorded. Cumulative concentration-response curves for the rate and tension responses to isoprenaline, orciprenaline, terbutaline and fenoterol were obtained before incubation of the tissues with isoprenaline (10^–6 M), the tissues were then washed during 1 h to remove isoprenaline before constructing a post-incubation curve to the same agonist. Incubation with isoprenaline for 4 h caused parallel rightwards shifts to the curves, but extending the incubation to 8 h caused additional depression of the rate (85.2 ± 5.4%) and tension (68.9 ± 2.3%) maxima. Incubation with isoprenaline for 4 h only shifted the curves for orciprenaline to the right but depressed the post-incubation maximum rate and tension responses to terbutaline (74.8 ± 1.5 and 33.8 ± 2.5%) and fenoterol (85.6 ± 5.5 and 76.0 ± 5.1 %). Thus incubation with isoprenaline for 4 h induced desensitization of the cardiac -adrenoceptor which was revealed as a loss in sensitivity to both isoprenaline and other -adrenoceptor agonists. The reduced maxima were attributed to a loss of -adrenoceptors and exhaustion of the receptor reserve. Those experiments displaying the reduced maxima were used for calculation of dissociation constants (K_A) values by the method of Furchgott (1966) for irreversible antagonism. The values for the right atria were 57 (19–170)nM, 29 (8.4–100)M and 13 (1.9–8.4)M and for the left atria, 89 (15–510)nM, 25 (9.9–64)M and 18 (7.7–42)M for isoprenaline (8h incubation), terbutaline and fenoterol respectively. Each value was significantly greater than the corresponding EC₅₀ value and calculation of the fractional receptor occupancies revealed a substantial receptor reserve in all cases except terbutaline on left atria. Calculation of relative efficacies (e_r) showed greater values for fenoterol (6.7 and 2.9) than isoprenaline in spite of its weaker potency and affinity. Finally, the K_A values were not significantly different for the rate and tension responses of each agonist, although each was rate selective. This indicates that the -adrenoceptors mediating these responses are identical and that the rate selectivity is due to better occupancy-response coupling. The validity of the method is discussed with regard to criticism of the null method in general and by comparison with affinity values from binding data.Send offprint requests to K. J. Broadley at the above address     

Biochemical and behavioural effects of isamoltane,a β-adrenoceptor antagonist with affinity for the 5-HT1B receptor of rat brain

Summary The biochemical and behavioural effects of isamoltane, a \-adrenoceptor and 5-HT_1B receptor antagonist that has higher affinity for 5-HT_1B receptors than for 5-HTIA receptors, on 5-HT neurotransmission in the rat brain were examined. In binding experiments isamoltane was found to be about five times more potent as a ligand for the 5-HT_1B receptor than for the 5-HT_1A receptor (K_i values 21 and 112 nmol/l, respectively). Isamoltane increased the K⁺-evoked overflow of ³H from ³H-5-HT loaded slices of rat occipital cortex at 0.1 mol/l, consistent with inhibition of the terminal 5HT autoreceptor. In vivo, isamoltane significantly increased the concentration of 5-hydroxyindoleacetic acid in hypothalamus and hippocampus indicating an increased 5-HT turnover with a maximal effect at 3 mg/kg s.c. A higher dose produced a less pronounced effect. This effect did not seem to be due to the -adrenoceptor blocking action of isamoltane since the -adrenoceptor antagonists, (–)-alprenolol, betaxolol or ICI 118,551 had no significant effects on 5-HT turnover at 5 mg/kg s.c. Isamoltane at 3 mg/kg s.c. induced the wet-dog shake response which was blocked by the tryptophan hydroxylase inhibitor p-chlorophenylalanine. In contrast, the same response induced by the 5-HT₂ receptor agonist quipazine was not blocked by pretreatment with p-chlorophenylalanine. The wet-dog shakes evoked by isamoltane and quipazine were blocked by ritanserin, which indicates that 5-HT₂ receptors are involved in their expression. These observations indicate that isamoltane, by inhibiting the terminal 5-HT autoreceptors, increased the synaptic concentration of 5-HT to a level that induced a behavioural response. Send of offprint requests to S. B. Ross at the above addressThe present results have been presented in part at the Second IUPHAR Satellite Meeting on Serotonin, Basel, Switzerland, July 11–13, 1990     

Enhancement of ADP-induced aggregation by 5-HT in rabbit platelets

目的：研究５ＨＴ对ＡＤＰ介导的血小板聚集反应的增强作用．方法：以透光法、图像法和受体结合法评价聚集反应、单细胞内钙和三磷酸肌醇的含量．结果：５ＨＴ（００３－３μｍｏｌ·Ｌ－１）浓度依赖性地引起ＰＲＰ的透光度降低（ＤＬＴ），电镜结果显示血小板变形的同时伴有颗粒中心化，无聚集和释放反应．Ｆｕｒａ２负载后，５ＨＴ升高［Ｃａ２＋］ｉ，９０秒达峰值，ＩＰ３一过性升高．ＡＤＰ同样引起ＤＬＴ，但可被５ＨＴ消除，呈浓度依赖性．ＡＤＰ的聚集反应和［Ｃａ２＋］ｉ动员则由于５ＨＴ预处理而升高．结论：５ＨＴ增强ＡＤＰ的聚集反应与５ＨＴ的细胞内钙动员及ＡＤＰ的外钙内流两者的叠加作用有关．     

Protective effects of compound FLZ,a novel synthetic analogue of squamosamide,on β-amyloid-induced rat brain mitochondrial dysfunction in vitro

Aim： The aim of the present study was to assess the effects of N-[2-（4-hydroxyphenyl）ethyl]-2-（2,5-dimethoxyphenyl）-3-（3- methoxy-4-hydroxyphenyl） acrylamide （compound FLZ）, a novel synthetic analogue of squamosamide, on the dysfunction of rat brain mitochondria induced by Aβ25-35 in vitro. Methods： Isolated rat brain mitochondria were incubated with aged Aβ25-35 for 30 rain in the presence and absence of FLZ （1-100 μmol/L）. The activities of.key mitochondrial enzymes, the production of hydrogen peroxide （H2O2） and superoxide anion （O2^-）, and the levels of glutathione （GSH） in mitochondria were examined. Mitochondrial swelling and the release of cytochrome c from mitochondria were assessed by biochemical and Western blot methods, respectively. Results： Incubation of mitochondria with aged Aβ25-35 inhibited the activities of α-ketoglutarate dehydrogenase （α-KGDH）, pyruvate dehydrogenase （PDH） and respiratory chain complex IV. It also resulted in increased H2O2 and O2^- production, and decreased the GSH level in mitochondria. Furthermore, it induced mitochondrial swelling and cytochrome c release from the mitochondria. The addition of FLZ （100 μmol/L） prior to treatment with Aβ25-35 significantly prevented these toxic effects of Aβ25-35 on the mitochondria. Conclusion： FLZ has a protective effect against Aβ25-35-induced mitochondrial dysfunction in vitro.     

Protective effects of compound FLZ,a novel synthetic analogue of squamosamide,on β-amyloid-induced rat brain mitochondrial dysfunction in vitro

Aim:

The aim of the present study was to assess the effects of N-[2-(4-hydroxyphenyl)ethyl]-2-(2,5-dimethoxyphenyl)-3-(3-methoxy-4-hydroxyphenyl) acrylamide (compound FLZ), a novel synthetic analogue of squamosamide, on the dysfunction of rat brain mitochondria induced by Aβ_25–35 in vitro.

Methods:

Isolated rat brain mitochondria were incubated with aged Aβ_25–35 for 30 min in the presence and absence of FLZ (1–100 μmol/L). The activities of key mitochondrial enzymes, the production of hydrogen peroxide (H₂O₂) and superoxide anion (O₂^·-), and the levels of glutathione (GSH) in mitochondria were examined. Mitochondrial swelling and the release of cytochrome c from mitochondria were assessed by biochemical and Western blot methods, respectively.

Results:

Incubation of mitochondria with aged Aβ_25–35 inhibited the activities of α-ketoglutarate dehydrogenase (α-KGDH), pyruvate dehydrogenase (PDH) and respiratory chain complex IV. It also resulted in increased H₂O₂ and O₂^·- production, and decreased the GSH level in mitochondria. Furthermore, it induced mitochondrial swelling and cytochrome c release from the mitochondria. The addition of FLZ (100 μmol/L) prior to treatment with Aβ_25–35 significantly prevented these toxic effects of Aβ_25–35 on the mitochondria.

Conclusion:

FLZ has a protective effect against Aβ_25–35-induced mitochondrial dysfunction in vitro.     

Effects of a hydroxylated metabolite of the β-adrenoreceptor antagonist,carvedilol, on post-ischaemic splanchnic tissue injury

1. Reactive oxygen species have been demonstrated to play a critical role in post-ischaemic tissue injury. The present experiment was designed to evaluate the effects of SB 211475, a hydroxylated metabolite of the new β-adrenoceptor antagonist, carvedilol, on rat splanchnic ischaemia (SI, 60 min) and reperfusion(R)-induced shock and tissue injury.
2. Administration of SB 211475 two min before R attenuated SI/R injury in a dose-dependent manner. At doses of 0.5 mg kg⁻¹ and 1.0 mg kg⁻¹, SB 211475 exerted significant anti-shock and endothelial protective effects, characterized by prolonged survival times, increased survival rates, attenuated increases in tissue myeloperoxidase activity and haematocrits, and preserved endothelium-dependent vasorelaxation.
3. Administration of 1 mg kg⁻¹ carvedilol attenuated shock-induced tissue injury and endothelial dysfunction. However, administration of 0.5 mg kg⁻¹ carvedilol had no protective effects on post-ischaemic tissue injury.
4. Previous studies have shown that SB 211475 has virtually no β-blocking activity but possesses more potent antioxidant activity than carvedilol. In the present study, SB 211475 exerted more potent protective effects than the parent compound, suggesting that this metabolite of carvedilol is superior to carvedilol with regard to its protection against post-ischaemia tissue injury.

     

Enhancement of nose-to-brain delivery of basic fibroblast growth factor for improving rat memory impairments induced by co-injection of β-amyloid and ibotenic acid into the bilateral hippocampus

Basic fibroblast growth factor (bFGF) delivery to the brain of animals appears to be an emerging potential therapeutic approach to neurodegenerative diseases, such as Alzheimer's disease (AD). The intranasal route of administration could provide an alternative to intracerebroventricular infusion. A nasal spray of bFGF had been developed previously and the objective of the present study was to investigate whether bFGF nasal spray could enhance brain uptake of bFGF and ameliorate memory impairment induced by co-injection of β-amyloid_25-35 and ibotenic acid into bilateral hippocampus of rats. The results of brain uptake study showed that the AUC_0-12 h of bFGF nasal spray in olfactory bulb, cerebrum, cerebellum and hippocampus was respectively 2.47, 2.38, 2.56 and 2.19 times that of intravenous bFGF solution, and 1.11, 1.95, 1.40 and 1.93 times that of intranasal bFGF solution, indicating that intranasal administration of bFGF nasal spray was an effective means of delivering bFGF to the brain, especially to cerebrum and hippocampus. In Morris water maze tasks, intravenous administration of bFGF solution at high dose (40 μg/kg) showed little improvement on spatial memory impairment. In contrast, bFGF solution of the same dose following intranasal administration could significantly ameliorate spatial memory impairment. bFGF nasal spray obviously improved spatial memory impairment even at a dose half (20 μg/kg) of bFGF solution, recovered their acetylcholinesterase and choline acetyltransferase activity to the sham control level, and alleviated neuronal degeneration in rat hippocampus, indicating neuroprotective effects on the central nerve system. In a word, bFGF nasal spray may be a new formulation of great potential for treating AD.     

Structural analysis by the comparative molecular field analysis method of the affinity of β-adrenoreceptor blocking agents for 5-HT1A and 5-HT1B receptors

The affinities of 17β-adrenoreceptor antagonists for 5-HT_1A and 5-HT_1B receptors were evaluated in binding assays. A large range of K_i values (2–10,000 nM) was observed and ortho or meta substitution of the aromatic ring carrying the amino chain was implicated in the high affinity K_i values, whereas para substitution elicited a dramatic drop in activity. These variations were analyzed with two molecular design tools: the active analogue approach (AAA) and the new 3D-QSAR (quantitative structure activity relationship) method, comparative molecular field analysis (CoMFA). The AAA method emphasized, by superimposition of selected conformations of the molecules, the favorable and unfavorable volumes implicated in the receptor recognition. CoMFA generated a linear expression between the biological data and the different values of electrostatic and steric fields surrounding the molecules. It predicted the values of selected molecules but also those of new molecules not included in the study. The excellent accuracy of the prediction revealed the potential of the method for the design of new compounds. CoMFA demonstrated the important contribution of steric parameters, evaluated at 92%, compared to the electrostatic field (evaluated at 8%) to explain the affinity for 5-HT_1A and 5-HT_1B receptors. This study emphasizes also the importance of the occupancy of a hydrophobic pocket in the receptor site located near the area interacting with the aromatic moiety, and subsequently its use for the design of new, potent, specific antagonists of 5-HT_1A and 5-HT_1B receptors.     

Inhibition by viozan of extravasation induced in rat trachea by capsaicin is mediated exclusively by β2-adrenoceptors

The mechanism by which 2(3H)-benzothiazolone, 4-hydroxy-7-[2-[[2-[[3-(2-phenylethoxy)propyl]-sulphonyl]ethyl]amino]ethyl]-monohydrochloride (AR-C68397AA; viozan), a dual dopamine D2/beta2-adrenoceptor agonist which has shown promise in the treatment of chronic obstructive pulmonary disease (COPD), inhibits the extravasation of plasma protein induced by capsaicin in the tracheas of Brown Norway rats has been re-evaluated. Viozan (10-30 microg/kg given intratracheally; i.t.) inhibited dose-dependently the extravasation of plasma protein tagged with Evans Blue into rat trachea induced by capsaicin (10 microg/kg i.t.). Similar effects were seen with the selective beta2-adrenoceptor agonist, salbutamol (3-10 microg/kg i.t.), but the selective dopamine D2 receptor agonist, quinagolide (10-30 microg/kg i.t.), was inactive. The effects of viozan and salbutamol were abolished by propranolol (3 mg/kg) given intraperitoneally (i.p.) but unaffected by sulpiride (3 mg/kg i.p.). Thus, in c,ontrast to claims in the literature, a functional response to dopamine D2 receptor activation in a preclinical model of oedema arising from sensory nerve fibre activation in the rat lung could not be demonstrated. Moreover, no qualitative difference could be demonstrated between the response to a dual D2/beta2-adrenoceptor agonist and a selective beta2-adrenoceptor agonist. The observations call into question whether a dual D2/beta2-adrenoceptor agonist such as viozan would bring added benefit over established selective beta2-adrenoceptor agonists in the therapy     

Inhibition of acetylcholine turnover in rat hippocampus by intraseptal injections of β-endorphin and morphine

Summary Intraseptal administration of morphine (70 nmol) or -endorphin (0.7 nmol) reduced the rate of acetylcholine (ACh) turnover (TR_ACh) in rat hippocampus but not in striatum or cortex. These intraseptal injections failed to modify the ACh content and did not elicit analgesia. Naltrexone (15 mol/kg, i.p.) completely antagonized the decrease of hippocampal TR_ACh elicited by the two opiate receptor agonists. Furthermore, intraseptal injections of naltrexone partially blocked the decrease in hippocampal TR_ACh induced by intraperitoneal administration of morphine (70 mol/kg, i.p.). These data suggest that opiate agonists decrease hippocampal TR_ACh by regulating septal cholinergic neurons, and that this effect is not associated with analgesia.     

Protection by dexamethasone of the functional desensitization to β2-adrenoceptor-mediated responses in human lung mast cells

1. The β-adrenoceptor agonist, isoprenaline, inhibited the IgE-mediated release of histamine from human lung mast cells (HLMC) in a dose-dependent manner. Maximal inhibitory effects were obtained with 0.1 μM isoprenaline. However, the inhibition of histamine release from HLMC by isoprenaline (0.1 μM) was highly variable ranging from 33 to 97% inhibition (mean, 59±3%, n=27).
2. Long-term (24 h) incubation of HLMC with isoprenaline led to a subsequent reduction in the ability of a second exposure of isoprenaline to inhibit IgE-mediated histamine release from HLMC. The impairment in the ability of isoprenaline (0.1 μM) to inhibit histamine release following desensitizing conditions (1 μM isoprenaline for 24 h) was highly variable amongst HLMC preparations ranging from essentially negligible levels of desensitization in some preparations to complete abrogation of the inhibitory response in others (mean, 65±6% desensitization, n=27).
3. The ability of HLMC to recover from desensitization was investigated. Following desensitizing conditions (1 μM isoprenaline for 24 h), HLMC were washed and incubated for 24 h in buffer and the effectiveness of isoprenaline (0.1 μM) to inhibit IgE-mediated histamine release from HLMC was assessed. The extent of recovery was highly variable with some HLMC preparations failing to recover and others displaying a complete restoration of responsiveness to isoprenaline (mean, 40±6% recovery, n=23).
4. The effects of the glucocorticoid, dexamethasone, were also investigated. Long-term (24–72 h) treatments with dexamethasone (0.1 μM) had no effect on IgE-mediated histamine release from HLMC. Additionally, long-term (24–72 h) treatments with dexamethasone (0.1 μM) had no effect on the effectiveness of isoprenaline to inhibit histamine release. However, long-term (24–72 h) treatments with dexamethasone (0.1 μM) protected against the functional desensitization induced by incubation (24 h) of HLMC with isoprenaline (1 μM). The protective effect was time-dependent and pretreatment of HLMC with dexamethasone for either 24, 48 or 72 h prevented desensitization by either 15±7, 19±5 or 51±10%, respectively (n=5–7).
5. HLMC preparations which were relatively refractory to isoprenaline even after withdrawal (24 h) from desensitizing conditions responded more effectively to isoprenaline (0.1 μM) if dexamethasone (0.1 μM) was also included during the recovery period (19±9% recovery after 24 h in buffer; 50±8% recovery after 24 h with dexamethasone, n=5).
6. These data indicate that the responses of different HLMC preparations to isoprenaline, the susceptibility of HLMC to desensitization and the ability of HLMC to recover from desensitizing conditions varies markedly. Dexamethasone, which itself has no direct effects on IgE-mediated histamine release from HLMC, protected HLMC from the functional desensitization to β-adrenoceptor agonists. Because β₂-adrenoceptor agonists and glucocorticoids are important in the therapeutic management of asthma and as the HLMC is probably important in certain types of asthma, these findings may have wider clinical implications.

     

Characterization of adenosine receptors in rat brain by (−)[3H]N6-phenylisopropyladenosine

Summary (–)N⁶-Phenylisopropyladenosine, a potent agonist in adenosine-responsive cellular systems, has been labeled with tritium to high specific activity (26 Ci/mmol) and used to identify adenosine binding sites in rat brain membranes. (–)[H³]N⁶-Phenylisopropyladenosine binding was studied by a vacuum filtration technique. The binding was rapid, rapidly reversible, dependent on pH and temperature and stereospecific since the (–)isomer of N⁶-phenylisopropyladenosine was 40-fold more potent than the (+)isomer in competition experiments. The stereospecific binding sites were saturable and bound 0.8 pmol of (–)N⁶-phenylisopropyladenosine per mg of membrane protein. The dissociation constant (K_D) of (–)N⁶-phenylisopropyladenosine for these sites was 5–12 nM as determined independently by saturation and kinetic binding studies. Endogeneous ligands seem to occupy the binding sites since pretreatment with adenosine deaminase increased the specific binding.Adenosine and several adenosine derivatives were studied for their ability to compete with (–)[³H]N⁶-phenylisopropyladenosine binding. (–)N⁶-Phenylisopropyladenosine-5-monophosphate, N⁶-phenyladenosine, N⁶-benzyladenosine, 2-chloroadenosine and adenosine were most potent in displacing the radioligand from its binding sites and the IC₅₀-values ranged from 0.3–7 M. Physiologically inactive compounds such as inosine, hypoxanthine, adenine and the ribose-modified analogues 2-deoxyadenosine and 2,5-dideoxyadenosine did not substantially inhibit binding at concentrations up to 100 M. The adenosine antagonists isobutylmethylxanthine (IC₅₀ 3.2 M), theophylline (IC₅₀ 7.6 M) and caffeine (IC₅₀ 99 M) competed for the binding sites of (–)[³H]N⁶-phenylisopropyladenosine in a manner which parallels their known pharmacological activity whereas other phosphodiesterase inhibitors were ineffective.The (–)[³H]N⁶-phenylisopropyladenosine binding sites in rat brain membranes appear to be equivalent to adenosine receptor sites on the cell surface which have recently been classified as R-site adenosine receptors.This work is dedicated in memory of Erik Westermann (1923–1978) and Klaus Stock (1931–1978) who first described the potent pharmacological effects of N⁶-phenylisopropyladenosine and stimulated much of the recent interest in adenosine receptor research     

Difference in sensitivity to alkaline phosphatase treatment between rat reticulocyte membranes in which β-adrenoceptor desensitization was induced by isoproterenol,dibutyryl cAMP and phorbol ester

The effect of alkaline phosphatase (3.1.3.1) on desensitization of β-adrenoceptor-responsive adenylate cyclase and the role of phosphorylation in desensitization were examined. Treatment of rat reticulocytes with isoproterenol, dibutyryl cAMP and tetradecanoyl phorbol acetate (TPA) caused the desensitization of β-adrenoceptor-coupled adenylate cyclase. When the membranes from dibutyryl cAMP- and TPA-desensitized cells were incubated with alkaline phosphatase for 60 min at 30°C, pH 8.0, the desensitization of isoproterenol-stimulated adenylate cyclase was markedly attenuated in both preparations. When the membranes from isoproterenol-desensitized cells were treated with alkaline phosphatase under the same conditions, the attenuation of the desensitization of alkaline phosphatase was less than in the case of treatment with dibutyryl cAMP or TPA. In other words, isoproterenol-induced desensitization was more resistant to alkaline phosphatase treatment. Isoproterenol- and dibutyryl cAMP-induced desensitization of NaF-stimulated adenylate cyclase were also attenuated by alkaline phosphatase treatment. Although the stability of the G_s-catalytic unit complex of adenylate cyclase was reduced by isoproterenol treatment, the reduction of stability was also decreased by alkaline phosphatase treatment.     

Regulation of gap-junction protein connexin 43 by β-adrenergic receptor stimulation in rat cardiomyocytes

Aim:

β-adrenergic receptor (β-AR) agonists are among the most potent factors regulating cardiac electrophysiological properties. Connexin 43 (Cx43), the predominant gap-junction protein in the heart, has an indispensable role in modulating cardiac electric activities by affecting gap-junction function. The present study investigates the effects of short-term stimulation of β-AR subtypes on Cx43 expression and gap junction intercellular communication (GJIC) function.

Methods:

The level of Cx43 expression in neonatal rat cardiomyocytes (NRCM) was detected by a Western blotting assay. The GJIC function was evaluated by scrape loading/dye transfer assay.

Results:

Stimulation of β-AR by the agonist isoproterenol for 5 min induces the up-regulation of nonphosphorylated Cx43 protein level, but not total Cx43. Selective β₂-AR inhibitor ICI 118551, but not β₁-AR inhibitor {"type":"entrez-protein","attrs":{"text":"CGP20712","term_id":"874704353","term_text":"CGP20712"}}CGP20712, could fully abolish the effect. Moreover, pretreatment with both protein kinase A inhibitor H89 and G_i protein inhibitor pertussis toxin also inhibited the isoproterenol-induced increase of nonphosphorylated Cx43 expression. Isoproterenol-induced up-regulation of nonphosphorylated Cx43 is accompanied with enhanced GJIC function.

Conclusion:

Taken together, β₂-AR stimulation increases the expression of nonphosphorylated Cx43, thereby enhancing the gating function of gap junctions in cardiac myocytes in both a protein kinase A- and G_i-dependent manner.