Effect of stress on the chronotropic and inotropic responses to ß-adrenergic agonists in isolated atria of KOß2 mice.

Altered sensitivity to the chronotropic and inotropic effects of catecholamines and reduction in ß1/ß2-adrenoceptor (ß1/ß2-AR) ratio were reported in failing and in senescent human heart, as well as in isolated atria and ventricle of rats submitted to stress. This was due to downregulation of ß1-AR with or without up-regulation of ß2-AR. AIMS: To investigate the stress-induced behavior of ß1-AR in the heart of mice expressing a non-functional ß2-AR subtype. The guiding hypothesis is that the absence of ß2-AR signaling will not affect the behavior of ß1-AR during stress and that those are independent processes. MATERIALS AND METHODS: The chronotropic and inotropic responses to ß-AR agonists in isolated atria of stressed mice expressing a non-functional ß2-AR were analyzed. The mRNA and protein expressions of ß1- and ß2-AR were also determined. KEY FINDINGS: No deaths were observed in mice under stress protocol. Atria of stressed mice displayed reduced sensitivity to isoprenaline compared to the controls, an effect that was abolished by the ß2- and ß1-AR antagonists 50 nM ICI118,551 and 300 nM CGP20712A, respectively. Sensitivity and maximum response to the ß-agonists dobutamine and salbutamol were not altered by stress or ICI118,551. The responses to dobutamine and salbutamol were prevented by CGP20712A. The expression of ß1-AR was reduced at protein levels. SIGNIFICANCE: Collectively, our data provide evidence that the cardiac ß2-AR is not essential for survival in a stressful situation and that the stress-induced reduction of ß1-AR expression was independent of the ß2-AR presence.

Beta-Blocker Type Effect on Substrate Oxidation during HIIE in Heart Failure Patients: Pilot Data.

The effect of third and second-generation type of beta-blocker on substrate oxidation especially during high-intensity exercises are scarce. The objective of the study is to explore differences of beta-blocker regimens (vasodilating vs. non-vasodilating beta-blockers) for substrate oxidation during in high-intensity intermittent exercise (HIIE) in chronic heart failure and reduced ejection fraction (HFrEF). Eighteen CHF males (58.8 ± 9 years), 8 under use of ß1 specific beta-blockers+alfa 1-blocker and 10 using ß1 non-specific beta-blockers, were randomly assigned to 4 different HIIE, in a cross-over design. The 4 protocols were: 30 seconds (A and B) or 90 seconds (C and D) at 100% peak power output, with passive (A and C) or active recovery (50% of PPO; B and D). Energy expenditure (EE; kcal/min), quantitative carbohydrate (CHO) and lipid oxidation (g/min) and qualitative (%) contribution were calculated. Two-way ANOVA and Bonferroni post-hoc test were used (p-value ≤ 0.05) to compare CHO and lipid oxidation at rest and at 10min. Total exercise time or EE did not show differences for beta-blocker use. The type of beta-blocker use showed impact in CHO (%) and lipid (g/min and %) for rest and 10 min, but absolute contribution of CHO (g/min) was different just at 10min (Interaction p = 0.029). Higher CHO oxidation was found in vasodilating beta-blockers when comparing to non-vasodilating. According to our pilot data, there is an effect of beta-blocker type on substrate oxidation during HIIE, but no influence on EE or exercise total time in HFrEF patients.

Population pharmacokinetics of carvedilol enantiomers and their metabolites in healthy subjects and type-2 diabetes patients.

Carvedilol, a drug available as a racemic mixture, is metabolised into hydroxyphenylcarvedilol (OHC) by CYP2D6 and O-desmethylcarvedilol (DMC) by CYP2C9 followed by conjugation to glucuronides. In contrast to other ß-adrenergic receptor antagonists, carvedilol does not induce insulin resistance or worsen glycaemic control in diabetic hypertensive patients. This study aims to investigate the implications of type 2 diabetes (T2DM) on the pharmacokinetics of carvedilol enantiomers using an integrated population pharmacokinetic modelling approach. In total, 14 T2DM patients with good glycaemic control receiving standard doses of metformin and glibenclamide were evaluated along with a control group of 13 healthy subjects. Serial blood samples were collected up to 24h after administration of a single 25mg dose of racemic carvedilol. A multicompartmental population pharmacokinetic model describing the enantioselective disposition of the parent compound, OHC and DMC was developed in NONMEM v7.2. Even though data are limited, it appears that despite inhibition of CYP2C9 due to long-term glibenclamide administration to T2DM patients, overall no differences are observed in the total clearance of carvedilol when compared to healthy subjects (43.1 vs. 45.9L/h for (S)-(-)-carvedilol and 29.0 vs. 33.1L/h for (R)-(+)-carvedilol). These results provide evidence of a compensatory mechanism for the inhibition of CYP2C9, with higher contribution of CYP2D6 activity to the elimination of carvedilol. Consequently, no dose adjustment is recommended for carvedilol in T2DM patients receiving glibenclamide and metformin.

Validation of a method for sedatives and ß-blockers determination in swine, bovine and equine kidney using liquid chromatography coupled with tandem mass spectrometry.

A method for the determination of five sedatives and 14 ß-blocker residues in swine, bovine and equine kidney was validated. Samples were extracted with acetonitrile and purified using dispersive solid phase extraction (d-SPE) with Celite 545 with subsequent analysis by LC-MS/MS. A simplified protocol was applied to validate the method scope extension to include new matrices. Parameters such as recovery, trueness, linearity (r2), relative standard deviation (RSD), decision limit (CCα) and method capability (CCß) were measured for the bovine and equine kidney matrices. The method was applied to the analysis of more than 300 real samples and is currently included in the Brazilian National Residue Control Plan.

Chiral analysis of carvedilol and its metabolites hydroxyphenyl carvedilol and O-desmethyl carvedilol in human plasma by liquid chromatography-tandem mass spectrometry: Application to a clinical pharmacokinetic study.

Carvedilol is an antihypertensive drug, which is available in clinical practice as a racemic mixture. (S)-(-)-carvedilol is a ß- and α1-adrenergic antagonist, while (R)-(+)-carvedilol only acts as an α1-adrenergic antagonist. Carvedilol is metabolized mainly by glucuronidation and, to a lesser extent, by CYP2D6 to hydroxyphenyl carvedilol (OHC) and by CYP2C9 to O-desmethyl carvedilol (DMC). This study describes the development and validation of a method for the sequential analysis of the enantiomers of carvedilol, OHC and DMC in plasma using a Chirobiotic(®) V chiral-phase column coupled to an LC-MS/MS system. The method was linear in the range of 0.05-100, 0.05-10 and 0.02-10 ng/mL for the carvedilol, OHC and DMC enantiomers, respectively. Application of the method to the investigation of a patient with type 2 diabetes mellitus treated with a single oral dose of 25mg racemic carvedilol showed plasma accumulation of the (R)-(+)-carvedilol, (R)-(+)-DMC and (R)-(+)-OHC enantiomers. These results suggest that plasma accumulation of (R)-(+)-carvedilol cannot be explained by its oxidative metabolism.

Influence of gestational diabetes mellitus on the stereoselective kinetic disposition and metabolism of labetalol in hypertensive patients.

PURPOSE: This study investigated the influence of gestational diabetes mellitus on the kinetic disposition and stereoselective metabolism of labetalol administered intravenously or orally. METHODS: Thirty hypertensive women during the last trimester of pregnancy were divided into four groups: non-diabetic and diabetic women treated with intravenous or oral labetalol. RESULTS: The pharmacokinetics of labetalol was not stereoselective in diabetic or non-diabetic pregnant women receiving the drug intravenously. However, oral administration of labetalol resulted in lower values of the area under the plasma concentration versus time curve (AUC) for the ß-blocker (RR) than for the other enantiomers in both diabetic and non-diabetic women. Gestational diabetes mellitus caused changes in the kinetic disposition of the labetalol stereoisomers when administered orally. The AUC values for the less potent adrenoceptor antagonist (SS) and for the α-blocking (SR) isomers were higher in diabetic than in non-diabetic pregnant women. CONCLUSIONS: The approximately 100% higher AUC values obtained for the (SR) isomer in diabetic pregnant women treated with oral labetalol may be of clinical relevance in terms of the α-blocking activity of this isomer.

É necessário suspender o betabloqueador na insuficiência cardíaca descompensada com baixo débito? / Is it necessary to suspend betablockers in decompensated heart failure with low output? / ¿Es necesario suspender el betabloqueante en la insuficiencia cardíaca descompensada con bajo débito?

FUNDAMENTO: Há evidências de que a suspensão do betabloqueador (BB) na descompensação cardíaca pode aumentar mortalidade. A dobutamina (dobuta) é o inotrópico mais utilizado na descompensação, no entanto, BB e dobuta atuam no mesmo receptor com ações antagônicas, e o uso concomitante dos dois fármacos poderia dificultar a compensação. OBJETIVO: Avaliar se a manutenção do BB associado à dobuta dificulta a compensação cardíaca. MÉTODOS: Estudados 44 pacientes com FEVE < 45 por cento e necessidade de inotrópico. Divididos em três grupos de acordo com o uso de BB. Grupo A (n=8): os que não usavam BB na admissão; Grupo B (n=25): os que usavam BB, porém foi suspenso para iniciar a dobuta; Grupo C (n=11): os que usaram BB concomitante à dobuta. Para comparação dos grupos, foram utilizados os testes t de Student, exato de Fisher e qui-quadrado. Considerado significante p < 0,05. RESULTADOS: FEVE média de 23,8 ± 6,6 por cento. O tempo médio do uso de dobuta foi semelhante nos três grupos (p=0,35), e o uso concomitante da dobuta com o BB não aumentou o tempo de internação (com BB 20,36 ± 11,04 dias vs sem BB 28,37 ± 12,76 dias, p=NS). Na alta, a dose do BB foi superior nos pacientes em que a medicação não foi suspensa (35,8 ± 16,8 mg/dia vs 23,0 ± 16,7 mg/dia, p=0,004). CONCLUSÃO: A manutenção do BB associado à dobuta não aumentou o tempo de internação e não foi acompanhada de pior evolução. Os pacientes que não suspenderam o BB tiveram alta com doses mais elevadas do medicamento.

BACKGROUND: There is evidence that the suspension of betablockers (BB) in decompensated heart failure may increase mortality. Dobutamine (dobuta) is the most commonly used inotrope in decompensation, however, BB and dobuta act with the same receptor with antagonist actions, and concurrent use of both drugs could hinder compensation. OBJECTIVE: To evaluate whether the maintenance of BB associated with dobuta difficults cardiac compensation. METHODS: We studied 44 patients with LVEF < 45 percent and the need for inotropics. Divided into three groups according to the use of BB. Group A (n=8): those who were not using BB at baseline; Group B (n=25): those who used BB, but was suspended to start dobuta; Group C (n = 11): those who used BB concomitant to dobuta. To compare groups, we used the Student t, Fisher exact and chi-square tests. Considered significant if p < 0.05. RESULTS: Mean LVEF 23.8 ± 6.6 percent. The average use of dobuta use was similar in all groups (p = 0.35), and concomitant use of dobutamine with BB did not increase the length of stay (BB 20.36 ± 11.04 days vs without BB 28.37 ± 12.76 days, p = NS). In the high dose, BB was higher in patients whose medication was not suspended (35.8 ± 16.8 mg/day vs 23.0 ± 16.7 mg/day, p = 0.004). CONCLUSION: Maintaining BB associated with dobutamine did not increase the length of hospitalization and was not associated with the worst outcome. Patients who did not suspend BB were discharged with higher doses of the drug.

FUNDAMENTO: Hay evidencias de que la suspensión del betabloqueante (BB) en la descompensación cardíaca puede aumentar la mortalidad. La dobutamina (dobuta) es el inotrópico más utilizado en la descompensación, mientras tanto, BB y dobuta actúan en el mismo receptor con acciones antagónicas, y el uso concomitante de los dos fármacos podría dificultar la compensación. OBJETIVO: Evaluar si la manutención del BB asociado a la dobuta dificulta la compensación cardíaca. MÉTODOS: Estudiados 44 pacientes con FEVI < 45 por ciento y necesidad de inotrópico. Divididos en tres grupos de acuerdo con el uso de BB. Grupo A (n=8): los que no usaban BB en la admisión; Grupo B (n=25): los que usaban BB, sin embargo fue suspendido para iniciar la dobuta; Grupo C (n=11): los que usaron BB concomitantemente a la dobuta. Para comparación de los grupos, fueron utilizados los test t de Student, exacto de Fisher y qui-cuadrado. Considerado significante P < 0,05. RESULTADOS: FEVI media de 23,8±6,6 por ciento. El tiempo medio de uso de dobuta fue semejante en los tres grupos (p=0,35), y el uso concomitante de la dobuta con el BB no aumentó el tiempo de internación (con BB 20,36 ± 11,04 días vs sin BB 28,37 ± 12,76 días, p=NS). En el alta, la dosis del BB fue superior en los pacientes en que la medicación no fue suspendida (35,8 ± 16,8 mg/día vs 23,0 ± 16,7 mg/día, p=0,004). CONCLUSIÓN: La manutención del BB asociado a la dobuta no aumentó el tiempo de internación y no fue acompañada de peor evolución. Los pacientes que no suspendieron el BB tuvieron alta con dosis más elevadas del medicamento.

[Is it necessary to suspend betablockers in decompensated heart failure with low output?]. / É necessário suspender o betabloqueador na insuficiência cardíaca descompensada com baixo débito?

BACKGROUND: there is evidence that the suspension of betablockers (BB) in decompensated heart failure may increase mortality. Dobutamine (dobuta) is the most commonly used inotrope in decompensation, however, BB and dobuta act with the same receptor with antagonist actions, and concurrent use of both drugs could hinder compensation. OBJECTIVE: to evaluate whether the maintenance of BB associated with dobuta difficults cardiac compensation. METHODS: we studied 44 patients with LVEF < 45% and the need for inotropics. Divided into three groups according to the use of BB. Group A (n=8): those who were not using BB at baseline; Group B (n=25): those who used BB, but was suspended to start dobuta; Group C (n = 11): those who used BB concomitant to dobuta. To compare groups, we used the Student t, Fisher exact and chi-square tests. Considered significant if p < 0.05. RESULTS: mean LVEF 23.8 ± 6.6%. The average use of dobuta use was similar in all groups (p = 0.35), and concomitant use of dobutamine with BB did not increase the length of stay (BB 20.36 ± 11.04 days vs without BB 28.37 ± 12.76 days, p = NS). In the high dose, BB was higher in patients whose medication was not suspended (35.8 ± 16.8 mg/day vs 23.0 ± 16.7 mg/day, p = 0.004). CONCLUSION: maintaining BB associated with dobutamine did not increase the length of hospitalization and was not associated with the worst outcome. Patients who did not suspend BB were discharged with higher doses of the drug.

Issues in drug metabolism of major antihypertensive drugs: beta-blockers, calcium channel antagonists and angiotensin receptor blockers.

Several first-line antihypertensive drugs, including calcium channel blockers, beta-adrenergic blockers and angiotensin receptor blockers, undergo metabolism through different CYP isoforms. As a consequence of CYP-dependent metabolism, wide interindividual variability of plasma concentrations of antihypertensive drugs has been found in clinical practice compromising blood pressure lowering response and clinical outcomes. Several factors, including aging, hepatic impairment, drug interactions, conditions affecting hepatic blood supply and polymorphisms, contribute to changes in oral and systemic clearance affecting drug exposure during antihypertensive therapy and cardiovascular response. Considering that the degree of blood pressure reduction is related to antihypertensive drug plasma concentrations, a greater knowledge of the sources of pharmacokinetic variability of hepatically eliminated antihypertensive drugs and the applicability of an individualized approach in hypertension management by means of pharmacokinetic/pharmacodynamic modeling and pharmacogenetic testing could enhance blood pressure lowering response to pharmacological therapy. The aim of the present review is to discuss the relevance of drug metabolism in the treatment of hypertension.

Central leptin action improves skeletal muscle AKT, AMPK, and PGC1 alpha activation by hypothalamic PI3K-dependent mechanism.

Central leptin action requires PI3K activity to modulate glucose homeostasis and peripheral metabolism. However, the mechanism behind this phenomenon is not clearly understood. We hypothesize that hypothalamic PI3K activity is important for the modulation of the AMP-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) pathway, PGC1 alpha, and AKT in skeletal muscle (SM). To address this issue, we injected leptin into the lateral ventricle of rats. Hypothalamic JAK2 and AKT were activated by intracerebroventricular (ICV) injection of leptin in a time-dependent manner. Central leptin improved tolerance to glucose (GTT), increased PGC1 alpha expression, and AKT, AMPK, ACC and JAK2 phosphorylation in the soleus muscle. Previous ICV administration of either LY294002 or propranolol (IP) blocked these effects. We concluded that the activation of the hypothalamic PI3K pathway is important for leptin-induced AKT phosphorylation, as well as for active catabolic pathway through AMPK and PGC1 alpha in SM. Thus, a defective leptin signalling PI3K pathway in the hypothalamus may contribute to peripheral resistance to insulin associated to diet-induced obesity.

Influence of quinidine, cimetidine, and ketoconazole on the enantioselective pharmacokinetics and metabolism of metoprolol in rats.

Metoprolol is a beta-blocker and its racemic mixture is used for the treatment of hypertension. In the present study we investigated the influence of CYP2D and CYP3A on the stereoselective metabolism of metoprolol in rats. Male Wistar rats (n = 6 per group) received racemic metoprolol (15 mg/kg) orally, with or without pretreatment with the CYP inhibitor ketoconazole (50 mg/kg), cimetidine (150 mg/kg), or quinidine (80 mg/kg). Blood samples were collected up to 48 h after metoprolol administration. The plasma concentrations of the stereoisomers of metoprolol, O-demethylmetoprolol (ODM), alpha-hydroxymetoprolol (OHM) (Chiralpak AD column), and metoprolol acidic metabolite (AODM) (Chiralcel OD-R column) were determined by HPLC using fluorescence detection (lambda(exc) = 229 nm; lambda(em) = 298 nm). CYP3A inhibition by ketoconazole reduced the plasma concentrations of ODM and AODM and favored the formation of OHM. CYP2D and CYP3A inhibition by cimetidine reduced the plasma concentrations of OHM and AODM and favored the formation of ODM. The inhibition of CYP2D by quinidine reduced the plasma concentrations of OHM and favored the formation of ODM. In conclusion, the results suggest that CYP3A is involved in the formation of ODM and CYP2D is involved in the formation of AODM.

Cellular and molecular abnormalities of a macronodular adrenal hyperplasia causing beta-blocker-sensitive Cushing's syndrome

Cushing's syndrome due to ACTH-independent macronodular adrenal hyperplasia (AIMAH) can be associated with abnormal responses of aberrantly expressed adrenocortical receptors. This study aimed to characterize in vitro the pathophysiology of hypercortisolism in a b-blocker-sensitive Cushing's syndrome due to AIMAH. Cortisol secretion profile under aberrant receptors stimulation revealed hyperresponsiveness to salbutamol (beta2-adrenoceptor agonist), cisapride (5-HT4 receptor agonist), and vasopressin in AIMAH cultured cells, but not in normal adrenocortical cells. By RT-PCR, AIMAH tissues revealed beta2-adrenoceptor overexpression rather than ectopical expression. MC2R expression was similar in both AIMAH and normal adrenocortical tissues. Curiously, cortisol levels of AIMAH cells under basal condition were 15-fold higher than those of control cells and were not responsive to ACTH. Analysis of culture medium from AIMAH cells could detect the presence of ACTH, which was immunohistochemically confirmed. Finally, the present study of AIMAH cells has identified: a) cortisol hyperresponsiveness to catecholamines, 5-HT4 and vasopressin in vitro, in agreement with clinical screening tests; b) abnormal expression of beta2-adrenoceptors in some areas of the hyperplastic adrenal tissue; c) autocrine loop of ACTH production. Altogether, the demonstration of aberrant responses to hormonal receptors and autocrine hormone production in the same tissue supports the assumption of multiple molecular alterations in adrenal macronodular hyperplasia.

A síndrome de Cushing secundária à hiperplasia adrenal macronodular independente de ACTH (AIMAH) pode estar associada com respostas anômalas a estímulos sobre receptores hormonais expressos de maneira aberrante no córtex adrenal. O objetivo deste trabalho foi caracterizar a fisiopatologia do hipercortisolismo in vitro na síndrome de Cushing responsiva a beta-bloqueadores decorrente de AIMAH. Em cultura de células, a secreção de cortisol apresentou resposta aumentada ao salbutamol (agonista beta2-adrenérgico), à cisaprida (agonista de receptor 5-HT4) e à vasopressina, na AIMAH mas não no córtex adrenal normal. O estudo de receptores aberrantes por RT-PCR demonstrou que o gene do receptor beta2-adrenérgico estava superexpresso (e não expresso ectopicamente) nos fragmentos da AIMAH quando comparado ao tecido normal. A expressão de MC2R foi semelhante em ambos. Curiosamente, o nível basal de secreção de cortisol pelas células da AIMAH foi 15 vezes superior às células normais, não havendo resposta das células AIMAH ao estímulo com ACTH. A análise do meio de cultura das células AIMAH revelou a presença de ACTH, que foi confirmada por estudo imuno-histoquímico. Em suma, este estudo demonstrou: a) aumento dos níveis de cortisol in vitro em resposta a catecolaminas, 5-HT4 e vasopressina, correspondendo aos resultados dos testes clínicos para pesquisa de receptores aberrantes; b) expressão anormal de receptores beta2-adrenérgicos em algumas áreas de hiperplasia; c) produção autócrina de ACTH. Estes resultados envolvendo ativação de receptores aberrantes e estímulo hormonal autócrino no mesmo tecido favorecem a hipótese da existência de alterações moleculares múltiplas na hiperplasia adrenal macronodular.

Cellular and molecular abnormalities of a macronodular adrenal hyperplasia causing beta-blocker-sensitive Cushing's syndrome.

Cushing's syndrome due to ACTH-independent macronodular adrenal hyperplasia (AIMAH) can be associated with abnormal responses of aberrantly expressed adrenocortical receptors. This study aimed to characterize in vitro the pathophysiology of hypercortisolism in a beta-blocker-sensitive Cushing's syndrome due to AIMAH. Cortisol secretion profile under aberrant receptors stimulation revealed hyperresponsiveness to salbutamol (beta2-adrenoceptor agonist), cisapride (5-HT4 receptor agonist), and vasopressin in AIMAH cultured cells, but not in normal adrenocortical cells. By RT-PCR, AIMAH tissues revealed beta2-adrenoceptor overexpression rather than ectopical expression. MC2R expression was similar in both AIMAH and normal adrenocortical tissues. Curiously, cortisol levels of AIMAH cells under basal condition were 15-fold higher than those of control cells and were not responsive to ACTH. Analysis of culture medium from AIMAH cells could detect the presence of ACTH, which was immunohistochemically confirmed. Finally, the present study of AIMAH cells has identified: a) cortisol hyperresponsiveness to catecholamines, 5-HT4 and vasopressin in vitro, in agreement with clinical screening tests; b) abnormal expression of beta2-adrenoceptors in some areas of the hyperplastic adrenal tissue; c) autocrine loop of ACTH production. Altogether, the demonstration of aberrant responses to hormonal receptors and autocrine hormone production in the same tissue supports the assumption of multiple molecular alterations in adrenal macronodular hyperplasia.

Stereoselective analysis of metoprolol and its metabolites in rat plasma with application to oxidative metabolism.

We investigated the stereoselective kinetic disposition and metabolism of metoprolol (MET) in rats. The racemic MET (15 mg/kg) was given by oral gavage and blood samples were collected from 0 to 10h (n=6 at each time point). The enantiomeric concentrations of MET and its metabolites alpha-hydroxymetoprolol (alpha-OHM) and O-demethylmetoprolol (ODM) were determined by HPLC using a Chiralpak AD chiral column and fluorescence detection. The pharmacokinetic parameters of unchanged MET and the formation of ODM did not show to be stereoselective. In contrast, the AUC (ng h/mL) of alpha-hydroxymetoprolol isomers were higher to I'R [638.2(525.2-706.2) for 1'R2R and 659.6(580.4-698.1) for 1'R,2S, mean, (95%CI)] than to I'S products [58.3(47.4-66.1) for 1'S,2R and 57.1(44.7-67.9) for 1'S,2S, mean, (95%CI)]. We conclude that the kinetic disposition of unchanged MET and the formation of ODM are not enantioselective in rats but the metabolism of alpha-OHM yields predominantly the 1'R-product.

Rapid eye movement sleep deprivation-induced down-regulation of beta-adrenergic receptors in the rat brainstem and hippocampus.

Rapid eye movement (REM) sleep deprivation induces a cortical down-regulation of beta-adrenergic receptors. Down-regulation of cortical beta-adrenergic receptors is consistently observed after a number of different chronic antidepressant treatments (drugs and electroconvulsive shock). REM sleep deprivation has an antidepressant effect in humans, and in rats, it decreases immobility in the behavioral despair test, an effect also produced by antidepressant treatments. To verify whether REM sleep deprivation also affects hippocampal beta-adrenergic receptors, we carried out the binding of [3H]-dihydroalprenolol ([3H]-DHA) to hippocampal membranes from rats deprived of REM sleep for 96 h. We also determined the binding of [3H]-DHA to brainstem membranes, a brain region where noradrenergic nuclei are located. Rats were deprived of REM sleep using a water tank with multiple small platforms. [3H-DHA] saturation conditions (concentrations ranging from 0.15 to 6 nM) were obtained in a crude hippocampus and brainstem membrane preparation. Nonspecific binding was determined using DL-propranolol in hippocampus homogenates. In the brainstem homogenates, nonspecific binding was determined in the presence of DL-propranolol or L-isoproterenol. The results obtained showed statistically significant down-regulation of beta-adrenergic receptors in both the hippocampus and the brainstem after REM sleep deprivation. In the hippocampus, there was also a significant decrease in the dissociation constant (KD). In the brainstem, a significant decrease in KD was observed when DL-propranolol was used to determine nonspecific binding. The down-regulation of beta-adrenergic receptors in the hippocampus and brainstem suggests the involvement of these brain areas in the antidepressant effect of REM sleep deprivation.

Altered expression of autonomic neurotransmitter receptors and proliferative responses in lymphocytes from a chronic mild stress model of depression: effects of fluoxetine.

We studied beta-adrenergic and muscarinic cholinergic receptor (MR) expression and proliferative response in lymphocytes from animals under chronic mild stress (CMS) model of depression (CMS animals). Animals were subjected to CMS (periods of food or water deprivation, changes in lighting conditions, tilted cage, etc.) for 12 weeks. CMS lymphocytes showed an altered mitogen-induced proliferation. CMS-B and -T lymphocytes showed an increment on beta-adrenoceptor number and on intracellular responses to a beta-agonist. CMS-T cells showed higher MR expression and lower cGMP responses than normal lymphocytes. MR were not detectable in normal B cells while CMS-B cells showed both MR expression and cGMP response. Beta and muscarinic stimulation influenced lymphocyte proliferative responses, in accordance with cAMP and cGMP responses. After 12 weeks of the CMS procedure, animals were treated with fluoxetine while the CMS procedure continued. Fluoxetine treatment reverted the alterations induced by CMS. These findings suggest a possible mechanism for the immune alterations found in depressive disorders and for the effect of fluoxetine treatment on immune response.

Daily changes in beta-adrenergic sensitivity of rat submandibular gland. Correlation with beta-adrenoceptor rhythm.

Neurons from the superior cervical ganglia (SCG) innervate the submandibular gland and release noradrenaline during the dark phase of the daily photoperiod. Since in the pineal, another structure innervated by sympathetic neurons, nocturnal activation of the SCG is associated with beta-adrenergic sub- and super-sensitivity rhythms, the possible existence of similar phenomena in the rat submandibular gland was assessed. Wistar female rats, kept on a 14:10 light/dark cycle (light from 06:00 to 20:00 h), were sacrificed at 09:00, 14:00, 20:00, 24:00 and 04:00 h. beta-Adrenoceptors were studied by 3H-dihydroalprenolol binding to membrane preparations. The equilibrium dissociation constant (Kd) did not change as a function of time while significant daily variations in maximal binding values (Bmax) were observed with a peak at 20:00 h. Changes in Bmax correlated with a high response of adenylate cyclase to isoproterenol. In addition, when the response in salivary flow to isoproterenol was measured. a shift to the left (about 1 logarithmic unit) in dose-response curves was observed at 19:00-20:00 has compared to 08:00-09:00 h. These daily variations in isoproterenol responsiveness seem not to depend on the pattern of eating since a 24-h starvation or a nocturnal starvation for 16-18 days did not abolish the morning-evening differences in the salivary flow response to isoproterenol. Rather, the results suggest that the daily variations in isoproterenol response correlate with beta-adrenergic super- and sub-sensitivity phenomena associated with the circadian release of noradrenaline from SCG neurons.