Benzodiazepine receptor ligand influences on learning: an endogenous modulatory mechanism mediated by benzodiazepines possibly of alimentary origin

In rats pre-but not post-training ip administration of either flumazenil, a central benzodiazepine (BSD) receptor antagonist, or of n-butyl-B-carboline-carboxylate (BCCB), an inverse agonist, enhanced retention of inhibitory avoidance learning. Flumazenil vlocked the enhancing effect of BCCB, and the inhibitory effect of the BZD agonists clonazepam and diazepam also given pre-training. Post-training administration of these drugs had no effects. The peripheral BZD receptor agonist/chloride channel blocker Ro5-4864 had no effect on the inhibitory avoidance task when given ip prior to training, buth it caused enhancement when given immediately post-training either ip or icv. This effect was blocked by PK11195, a competitive antagonist of Ro5-4864. These results suggest that ther is an endogenous mechanism mediated by BZD agonists, which is sensitive to inverse agonists and that normally down-regulates the formation of memories through a mechanism involving GABA-A receptors and the corresponding chloride channels. The most likely agonists for the endogenous mechanism suggested are the diazepam-like BZDs found in brain whose origin is possibly alimentary. Levels of these BZDs in the cortex were found to sharply decrease after inhibitory acoidance training or mere exposure to the training apparatus.

Glucose competence of the hepatoportal vein sensor requires the presence of an activated glucagon-like peptide-1 receptor.

Activation of the hepatoportal glucose sensors by portal glucose infusion leads to increased glucose clearance and induction of hypoglycemia. Here, we investigated whether glucagon-like peptide-1 (GLP-1) could modulate the activity of these sensors. Mice were therefore infused with saline (S-mice) or glucose (P-mice) through the portal vein at a rate of 25 mg/kg. min. In P-mice, glucose clearance increased to 67.5 +/- 3.7 mg/kg. min as compared with 24.1 +/- 1.5 mg/kg. min in S-mice, and glycemia decreased from 5.0 +/- 0.1 to 3.3 +/- 0.1 mmol/l at the end of the 3-h infusion period. Coinfusion of GLP-1 with glucose into the portal vein at a rate of 5 pmol/kg. min (P-GLP-1 mice) did not increase the glucose clearance rate (57.4 +/- 5.0 ml/kg. min) and hypoglycemia (3.8 +/- 0.1 mmol/l) observed in P-mice. In contrast, coinfusion of glucose and the GLP-1 receptor antagonist exendin-(9-39) into the portal vein at a rate of 0.5 pmol/kg. min (P-Ex mice) reduced glucose clearance to 36.1 +/- 2.6 ml/kg. min and transiently increased glycemia to 9.2 +/- 0.3 mmol/l at 60 min of infusion before it returned to the fasting level (5.6 +/- 0.3 mmol/l) at 3 h. When glucose and exendin-(9-39) were infused through the portal and femoral veins, respectively, glucose clearance increased to 70.0 +/- 4.6 ml/kg. min and glycemia decreased to 3.1 +/- 0.1 mmol/l, indicating that exendin-(9-39) has an effect only when infused into the portal vein. Finally, portal vein infusion of glucose in GLP-1 receptor(-/-) mice failed to increase the glucose clearance rate (26.7 +/- 2.9 ml/kg. min). Glycemia increased to 8.5 +/- 0.5 mmol/l at 60 min and remained elevated until the end of the glucose infusion (8.2 +/- 0.4 mmol/l). Together, our data show that the GLP-1 receptor is part of the hepatoportal glucose sensor and that basal fasting levels of GLP-1 sufficiently activate the receptor to confer maximum glucose competence to the sensor. These data demonstrate an important extrapancreatic effect of GLP-1 in the control of glucose homeostasis.

The Role of Skin Opioid Receptor System in Itch

Opioid receptors are key players in induction of chronic itch. This could be confirmed using opiate receptor knockout mice experiments and clinical studies on patients with chronic itch. We have induced a dry skin dermatitis as a model for chronic itching on -(MOR) and -(KOR) opioid receptor knockout (KO) mice. MOR KO mice scratched significantly less than wild type (WT). Additionally the epidermal hypertrophy caused by chronic dermatitis and the amount of epidermal nerve endings in MOR KO mice were significantly decreased than in WT mice. KOR KO mice showed similar scratching behavior as MOR KO mice; however the changes were less significant. In addition, we performed a double blind, placebo controlled, cross over study using topically applied opioid receptor antagonist, Naltrexone, on patients with pruritus in atopic dermatitis. The results revealed significant effects of the topical application of Naltrexone in patients with chronic pruritus (45% improvement of pruritus by VAS compared to placebo, n=24), but not in patients with acute pruritus (7%, n=15). These studies establish the clinical relevance of MOR system and the peripheral, epidermal nerve endings in chronic pruritus and warrant further research and therapeutic potential for such research.

Ampakine CX546 bolsters energetic response of astrocytes: a novel target for cognitive-enhancing drugs acting as alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor modulators.

Glutamate was previously shown to enhance aerobic glycolysis i.e. increase glucose utilization and lactate production with no change in oxygen levels, in mouse cortical astrocytes by a mechanism involving glutamate uptake. It is reported here that a similar response is produced in both hippocampal and cerebellar astrocytes. Application of the cognitive-enhancing drug CX546 promoted further enhancement of glucose utilization by astrocytes from each brain area following glutamate exposure. alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors represent the purported molecular target of cognitive-enhancing drugs such as CX546, and the presence of AMPA receptor subunits GluR1-4 was evidenced in astrocytes from all three regions by immunocytochemistry. AMPA itself did not stimulate aerobic glycolysis, but in the presence of CX546, a strong enhancement of glucose utilization and lactate production was obtained in cortical, hippocampal and cerebellar astrocytes. The effect of CX546 was concentration-dependent, with an EC(50) of 93.2 microm in cortical astrocytes. AMPA-induced glucose utilization in the presence of CX546 was prevented by the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the negative modulator GYKI 52466. In addition, the metabolic effect of CX546 in the presence of AMPA was mimicked by the AMPA receptor modulator cyclothiazide. Our data suggest that astrocyte energetics represents a novel target for cognitive-enhancing drugs acting as AMPA receptor modulators.

Activation of GPER-1 estradiol receptor downregulates production of testosterone in isolated rat Leydig cells and adult human testis.

PURPOSE: Estradiol (E2) modulates testicular functions including steroidogenesis, but the mechanisms of E2 signaling in human testis are poorly understood. GPER-1 (GPR30), a G protein-coupled membrane receptor, mediates rapid genomic and non-genomic response to estrogens. The aim of this study was to evaluate GPER-1 expression in the testis, and its role in estradiol dependent regulation of steroidogenesis in isolated rat Leydig cells and human testis. MATERIALS AND METHODS: Isolated Leydig cells (LC) from adult rats and human testicular tissue were used in this study. Expression and localization studies of GPER-1 were performed with qRT-PCR, immunofluorescence, immunohistochemistry and Western Blot. Luteinizing Hormone (LH) -stimulated, isolated LC were incubated with estradiol, G-1 (GPER-1-selective agonist), and estrogen receptor antagonist ICI 182,780. Testosterone production was measured with radioimmunoassay. LC viability after incubation with G-1 was measured using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. RESULTS: GPER-1 mRNA is abundantly expressed in rat LC and human testis. Co-localization experiments showed high expression levels of GPER-1 protein in LC. E2-dependent activation of GPER-1 lowers testosterone production in isolated rats LCs and in human testis, with statistically and clinically significant drops in testosterone production by 20-30% as compared to estradiol-naïve LC. The exposure to G-1 does not affect viability of isolated LCs. CONCLUSIONS: Our results indicate that activation of GPER-1 lowers testosterone levels in the rat and human testis. The expression of GPER-1 in human testis, which lack ERα, makes it an exciting target for developing new agents affecting testosterone production in men.

A constitutively active mutant of the alpha 1B-adrenergic receptor can cause greater agonist-dependent down-regulation of the G-proteins G9 alpha and G11 alpha than the wild-type receptor.

Rat 1 fibroblasts transfected to express either the wild-type hamster alpha 1B-adrenergic receptor or a constitutively active mutant (CAM) form of this receptor resulting from the alteration of amino acid residues 288-294 to encode the equivalent region of the human beta 2-adrenergic receptor were examined. The basal level of inositol phosphate generation in cells expressing the CAM alpha 1B-adrenergic receptor was greater than for the wild-type receptor, The addition of maximally effective concentrations of phenylephrine or noradrenaline resulted in substantially greater levels of inositol phosphate generation by the CAM alpha 1B-adrenergic receptor, although this receptor was expressed at lower steady-state levels than the wild-type receptor. The potency of both phenylephrine and noradrenaline to stimulate inositol phosphate production was approx. 200-fold greater at the CAM alpha 1B-adrenergic receptor than at the wild-type receptor. In contrast, endothelin 1, acting at the endogenously expressed endothelin ETA, receptor, displayed similar potency and maximal effects in the two cell lines. The sustained presence of phenylephrine resulted in down-regulation of the alpha subunits of the phosphoinositidase C-linked, pertussis toxin-insensitive, G-proteins G9 and G11 in cells expressing either the wild-type or the CAM alpha 1B-adrenergic receptor. The degree of down-regulation achieved was substantially greater in cells expressing the CAM alpha 1B-adrenergic receptor at all concentrations of the agonist. However, in this assay phenylephrine displayed only a slightly greater potency at the CAM alpha 1B-adrenergic receptor than at the wild-type receptor. There were no detectable differences in the basal rate of G9 alpha/G11 alpha degradation between cells expressing the wild-type or the CAMalpha 1B-adrenergic receptor. In both cell lines the addition of phenylephrine substantially increased the rate of degradation of these G-proteins, with a greater effect at the CAM alpha 1B-adrenergic receptor. The enhanced capacity of agonist both to stimulate second-messenger production at the CAM alpha 1B-adrenergic receptor and to regulate cellular levels of its associated G-proteins by stimulating their rate of degradation is indicative of an enhanced stoichiometry of coupling of this form of the receptor to G9 and G11.

Renal effects of adenosine A1-receptor blockade with 8-cyclopentyl-1,3-dipropylxanthine in hypoxemic newborn rabbits.

The key role of intrarenal adenosine in mediating the hypoxemic acute renal insufficiency in newborn rabbits has been well demonstrated using the nonspecific adenosine antagonist theophylline. The present study was designed to define the role of adenosine A1 receptors during systemic hypoxemia by using the specific A1-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). Renal function parameters were assessed in 31 anesthetized and mechanically ventilated newborn rabbits. In normoxia, DPCPX infusion induced a significant increase in diuresis (+44%) and GFR (+19%), despite a significant decrease in renal blood flow (RBF) (-22%) and an increase in renal vascular resistance (RVR) (+37%). In hypoxemic conditions, diuresis (-19%), GFR (-26%), and RBF (-35%) were decreased, whereas RVR increased (+33%). DPCPX administration hindered the hypoxemia-induced decrease in GFR and diuresis. However, RBF was still significantly decreased (-27%), whereas RVR increased (+22%). In all groups, the filtration fraction increased significantly. The overall results support the hypothesis that, in physiologic conditions, intrarenal adenosine plays a key role in regulating glomerular filtration in the neonatal period through preferential A1-mediated afferent vasoconstriction. During a hypoxemic stress, the A1-specific antagonist DPCPX only partially prevented the hypoxemia-induced changes, as illustrated by the elevated RVR and drop in RBF. These findings imply that the contribution of intrarenal adenosine to the acute adverse effects of hypoxemia might not be solely mediated via the A1 receptor.

Renal endothelin receptor type B upregulation in rats with low or high renin hypertension.

OBJECTIVES: To evaluate the role of endothelin-1 (ET-1) in hypertension, we investigated density and distribution of ETA and ETB receptors in hearts and kidneys of deoxycorticosterone acetate (DOCA)-salt and 1 kidney -- 1 clip (1K1C) hypertensive rats. METHODS: Five groups of uninephrectomized Wistar rats were put on a low salt diet. Three groups of rats drank tap water and two groups received saline. One group of each regimen received DOCA subcutaneously and two corresponding groups without DOCA served as controls. The fifth group of rats had the renal artery clipped to induce 1K1C hypertension. At 6 weeks, mean arterial pressure (MAP) was recorded and membrane binding assays using 125I-ET-1 were carried out. RESULTS: MAP was increased from control 122 +/- 3 to 155 +/- 6 and 218 +/- 11 mmHg in DOCA-salt and 1K1C rats, respectively, and cardiac weight index was increased. ETA receptors were predominantly expressed in the heart, whereas ETB receptors were predominant in the kidney. In the kidneys, the density of the ETB receptor subtype was upregulated in DOCA-salt and 1K1C rats from 160 +/- 8 to 217 +/- 12 and 190 +/- 2 fmol/mg (P < 0.05), respectively, and ETA tended to be downregulated (P = 0.057). Plasma renin activity was decreased in DOCA-salt rats from 17 +/- 3 to 0.17 +/- 0.01 ng/ml per h and increased in 1K1C rats on low salt diet to 30 +/- 5 ng/ml per h. CONCLUSIONS: Since ETB is the predominant endothelin receptor in the kidneys, upregulation of the ETB receptor mediating vasodilation and downregulation of the ETA receptor mediating vasoconstriction would be compatible with a mainly renal counter-regulatory effect of endothelin-1 to hypertension. Both low and high renin models of hypertension may be affected.

Effect of indomethacin on the renal response to angiotensin II receptor blockade in healthy subjects.

BACKGROUND: Non-steroidal anti-inflammatory drugs are known to promote sodium retention and to blunt the blood pressure lowering effects of several classes of antihypertensive agents including beta-blockers, diuretics and angiotensin converting enzyme (ACE) inhibitors. The purpose of the present study was to investigate the acute and sustained effects of indomethacin on the renal response to the angiotensin II receptor antagonist valsartan and to the ACE inhibitor enalapril. METHODS: Twenty normotensive subjects maintained on fixed sodium intake (100 mmol sodium/day) were randomized to receive for one week: valsartan 80 mg o.d., enalapril 20 mg o.d., valsartan 80 mg o.d. + indomethacin 50 mg bid and enalapril 20 mg o.d. + indomethacin 50 mg bid. This single-blind study was designed as a parallel (valsartan vs. enalapril) and cross-over trial (valsartan or enalapril vs. valsartan + indomethacin or enalapril + indomethacin). Renal hemodynamics and urinary electrolyte excretion were measured for six hours after the first and seventh administration of each treatment regimen. RESULTS: The results show that valsartan and enalapril have comparable renal effects characterized by no change in glomerular filtration rate and significant increases in renal plasma flow and sodium excretion. The valsartan- and enalapril-induced renal vasodilation is not significantly blunted by indomethacin. However, indomethacin similarly abolishes the natriuresis induced by the angiotensin II antagonist and the ACE inhibitor. CONCLUSIONS: This observation suggests that although angiotensin receptor antagonists do not affect prostaglandin metabolism, the administration of a non-steroidal anti-inflammatory drug blunts the natriuretic response to angiotensin receptor blockade.

Salt-dependent renal effects of an angiotensin II antagonist in healthy subjects.

This study was designed to evaluate in healthy volunteers the renal hemodynamic and tubular effects of the orally active angiotensin II receptor antagonist losartan (DuP 753 or MK 954). Losartan or a placebo was administered to 23 subjects maintained on a high-sodium (200 mmol/d) or a low-sodium (50 mmol/d) diet in a randomized, double-blind, crossover study. The two 6-day diet periods were separated by a 5-day washout period. On day 6, the subjects were water loaded, and blood pressure, renal hemodynamics, and urinary electrolyte excretion were measured for 6 hours after a single 100-mg oral dose of losartan (n = 16) or placebo (n = 7). Losartan induced no significant changes in blood pressure, glomerular filtration rate, or renal blood flow in these water-loaded subjects, whatever the sodium diet. In subjects on a low-salt diet, losartan markedly increased urinary sodium excretion from 115 +/- 9 to 207 +/- 21 mumol/min (P < .05). The fractional excretion of endogenous lithium was unchanged, suggesting no effect of losartan on the early proximal tubule in our experimental conditions. Losartan also increased urine flow rate (from 10.5 +/- 0.4 to 13.1 +/- 0.6 mL/min, P < .05); urinary potassium excretion (from 117 +/- 6.9 to 155 +/- 11 mumol/min); and the excretion of chloride, magnesium, calcium, and phosphate. In subjects on a high-salt diet, similar effects of losartan were observed, but the changes induced by the angiotensin II antagonist did not reach statistical significance. In addition, losartan demonstrated significant uricosuric properties with both sodium diets.(ABSTRACT TRUNCATED AT 250 WORDS)

Mineralocorticoid Receptor Antagonism in the Treatment of Chronic Central Serous Chorioretinopathy : A Pilot Study.

PURPOSE:: Based on experimental data showing that central serous chorioretinopathy could result from overactivation of mineralocorticoid receptor pathway in choroid vessels, the authors studied eplerenone, a mineralocorticoid receptor antagonist, as a potential treatment for chronic central serous chorioretinopathy. METHODS:: This nonrandomized pilot study included 13 patients with central serous chorioretinopathy of at least 4-month duration, treated with 25 mg/day of oral eplerenone for a week followed by 50 mg/day for 1 or 3 months. The primary outcome measure was the changes in central macular thickness recorded by optical coherence tomography, and the secondary outcomes included changes in foveal subretinal fluid (SRF) measured by OCT, in best-corrected visual acuity (BCVA) and the percentage of eyes achieving complete resolution of subretinal fluid during the treatment period. RESULTS:: Central macular thickness decreased significantly from 352 ± 139 μm at baseline to 246 ± 113 μm and 189 ± 99 μm at 1 and 3 months under eplerenone treatment (P < 0.05 and P < 0.01, respectively). At 3 months, the subretinal fluid significantly decreased compared with baseline subretinal fluid (P < 0.01) and best-corrected visual acuity significantly improved compared with baseline best-corrected visual acuity (P < 0.001). CONCLUSION:: Eplerenone treatment was associated with a significant reduction in central macular thickness, subretinal fluid level, and an improvement in visual acuity. Randomized controlled trials are needed to confirm these encouraging results.

Novel action of a wake-promoting neuropeptide in hippocampus : inhibition of nmda receptor function at excitatory synapses through orexin-a

One of the most intriguing functions of the brain is the ability to learn and memorize. The mechanism through which memory and learning are expressed requires the activation of NMDA receptors (NMDARs). These molecular entities are placed at the postsynaptic density of excitatory synapses and their function is tightly controlled by the actions of several modulators at the extracellular, intracellular and pore sites. A large part of the intracellular modulation comes from the action of G-protein coupled receptors (GPCRs). Through intracellular cascades typically involving kinases and phosphatases, GPCRs potentiate or inhibit NMDARs, controlling the conductive state but also the trafficking within the synapse. The GPCRs are involved in the modulation of a variety of brain functions. Many of them control cognition, memory and learning performance, therefore, their effects on NMDARs are extensively studied. The orexinergic system signals through GPCRs and it is well known for the regulation of waking, feeding, reward and autonomic functions. Moreover, it is involved in potentiating hippocampus-related cognitive tasks. Orexin receptors and fibers are present within the hippocampus, but whether these directly modulate hippocampal cells and synapses has not yet been determined. During my thesis, I studied orexinergic actions on excitatory synaptic transmission via whole-cell patch-clamp recordings in rat acute hippocampal slices. I observed that exogenously applied orexin-A (ox-A) exerted a strong inhibitory action on NMDAR-mediated synaptic potentials at mossy fiber (MF)-CA3 synapses, by postsynaptically activating orexin-2 receptors, a minor inhibition at Schaffer collateral-CAl synapses and did not affect other synapses with the CA3 area. Moreover, I demonstrated that the susceptibility of NMDARs to ox- A depends on the tone of endogenous orexin known to fluctuate during the day-night cycle. In fact, in slices prepared during the active period of the rats, when endogenous orexin levels are high, NMDAR-currents were not affected by exogenously applied ox-A. The inhibitory effect of ox-A was, however, reverted when interfering with the orexinergic system through intraperitoneal injections of almorexant, a dual orexin receptor antagonist, during the active phase prior to slice preparation. This thesis work suggests that the orexinergic system regulates NMDAR-dependent information flow through select hippocampal pathways depending on the time-of-day. The specific orexinergic modulation of NMDARs at MFs dampens the excitability of the hippocampal circuit and could impede the mechanisms related to memory formation, possibly also following extended periods of waking. -- La capacité d'apprentissage et de mémorisation est une des fonctions les plus intrigantes de notre cerveau. Il a été montré qu'elles requièrent l'activation des récepteurs NMDA (NMDARs). Ces entités moléculaires sont présentes au niveau de la densité post-synaptique des synapses excitatrices et leur fonction est étroitement contrôlée par l'action de nombreux modulateurs au niveau extracellulaire, intracellulaire et membranaire de ces récepteurs. Une grande partie de la modulation intracellulaire s'effectue via l'action de récepteurs couplés aux protéines G (GPCRs). Grace à leurs cascades intracellulaires typiquement impliquant des kinases et des phosphatases, les GPCRs favorisent l'activation ou l'inhibition des NMDARs, contrôlant ainsi leur perméabilité mais aussi leur mouvement à la synapse. Les GPCRs sont impliquées dans de nombreuses fonctions cérébrales telles que la cognition, la mémoire ainsi que la capacité d'apprentissage c'est pour cela que leurs effets sur les NMDARs sont très étudiés. Le système orexinergique fait intervenir ces GPCRs et est connu par son rôle dans la régulation de fonctions physiologiques telles que l'éveil, la prise alimentaire, la récompense ainsi que d'autres fonctions du système nerveux autonome. De plus, ce système est impliqué dans la régulation de tâches cognitives liées à l'hippocampe. Bien que les fibres et les récepteurs à l'orexine soient présents dans l'hippocampe, leur mécanisme d'action sur les cellules et les synapses de l'hippocampe n'a pas encore été élucidé. Durant ma thèse, je me suis intéressée aux effets de l'orexine sur la transmission synaptique excitatrice en utilisant la méthode d'enregistrement en patch-clamp en configuration cellule entière sur des tranches aiguës d'hippocampes de rats. J'ai observé que l'application exogène d'orexine A d'une part inhibe fortement les courants synaptiques dépendants de l'activation des NMDARs au niveau de la synapse entre les fibres moussues et CA3 via l'activation post-synaptique des orexine récepteurs 2 mais d'autre part n'inhibe que de façon mineure la synapse entre les collatérales de Schaffer et CAI et n'affecte pas les autres synapses impliquant CA3. J'ai également démontré que la sensibilité des NMDARs à l'orexine A dépend de sa concentration endogène qui fluctue durant le cycle éveil-sommeil. En effet, lorsque les coupes d'hippocampes sont préparées durant la période active de l'animal correspondant à un niveau endogène d'orexine élevé, l'application exogène d'orexine A n'a aucun effet sur les courants dépendants de l'activation des NMDARs. Cependant, l'injection dans le péritoine, durant la phase active de l'animal, d'un antagoniste des orexine récepteurs, l'almorexant, va supprimer l'effet inhibiteur de l'orexine A. Les résultats de ma thèse suggèrent donc que le système orexinergique module les informations véhiculées par les NMDARs via des voies de signalisation sélectives de l'hippocampe en fonction du moment de la journée. La modulation orexinergique des NMDARs au niveau des fibres moussues diminue ainsi l'excitabilité du circuit hippocampal et pourrait entraver les mécanismes liés à la formation de la mémoire, potentiellement après de longues périodes d'éveil.

Role of endothelin receptor subtypes in volume-stimulated ANF secretion.

The role of endothelin (ET) receptors was tested in volume-stimulated atrial natriuretic factor (ANF) secretion in conscious rats. Mean ANF responses to slow infusions (3 x 3.3 ml/8 min) were dose dependently reduced (P < 0.05) by bosentan (nonselective ET-receptor antagonist) from 64.1 +/- 18.1 (SE) pg/ml (control) to 52.6 +/- 16.1 (0.033 mg bosentan/rat), 16.1 +/- 7.6 (0. 33 mg/rat), and 11.6 +/- 6.5 pg/ml (3.3 mg/rat). The ET-A-receptor antagonist BQ-123 (1 mg/rat) had no effect relative to DMSO controls, whereas the putative ET-B antagonist IRL-1038 (0.1 mg/rat) abolished the response. In a second protocol, BQ-123 (>/=0.5 mg/rat) nonsignificantly reduced the peak ANF response (106.1 +/- 23.0 pg/ml) to 74.0 +/- 20.5 pg/ml for slow infusions (3.5 ml/8.5 min) but reduced the peak response (425.3 +/- 58.1 pg/ml) for fast infusions (6.6 ml/1 min) by 49.9% (P < 0.001) and for 340 pmoles ET-1 (328.8 +/- 69.5 pg/ml) by 83.5% (P < 0.0001). BQ-123 abolished the ET-1-induced increase in arterial pressure (21.8 +/- 5.2 mmHg at 1 min). Changes in central venous pressure were similar for DMSO and BQ-123 (slow: 0.91 and 1.14 mmHg; fast: 4.50 and 4.13 mmHg). The results suggest 1) ET-B receptors mainly mediate the ANF secretion to slow volume expansions of <1.6%/min; and 2) ET-A receptors mainly mediate the ANF response to acute volume overloads.

Acute AT1 receptor blockade does not improve the depressed baroreflex in rats with chronic renal hypertension

To assess the role of angiotensin II in the sensitivity of the baroreflex control of heart rate (HR) in normotensive rats (N = 6) and chronically hypertensive rats (1K1C, 2 months, N = 7), reflex changes of HR were evaluated before and after (15 min) the administration of a selective angiotensin II receptor antagonist (losartan, 10 mg/kg, iv). Baseline values of mean arterial pressure (MAP) were higher in hypertensive rats (195 ± 6 mmHg) than in normotensive rats (110 ± 2 mmHg). Losartan administration promoted a decrease in MAP only in hypertensive rats (16%), with no changes in HR. During the control period, the sensitivity of the bradycardic and tachycardic responses to acute MAP changes were depressed in hypertensive rats (~70% and ~65%, respectively) and remained unchanged after losartan administration. Plasma renin activity was similar in the two groups. The present study demonstrates that acute blockade of AT1 receptors with losartan lowers the MAP in chronic renal hypertensive rats without reversal of baroreflex hyposensitivity, suggesting that the impairment of baroreflex control of HR is not dependent on an increased angiotensin II level.

Adenosine A1 receptor-mediated inhibition of in vitro prolactin secretion from the rat anterior pituitary

In previous studies, we demonstrated biphasic purinergic effects on prolactin (PRL) secretion stimulated by an adenosine A2 agonist. In the present study, we investigated the role of the activation of adenosine A1 receptors by (R)-N6-(2-phenylisopropyl)adenosine (R-PIA) at the pituitary level in in vitro PRL secretion. Hemipituitaries (one per cuvette in five replicates) from adult male rats were incubated. Administration of R-PIA (0.001, 0.01, 0.1, 1, and 10 µM) induced a reduction of PRL secretion into the medium in a U-shaped dose-response curve. The maximal reduction was obtained with 0.1 µM R-PIA (mean ± SEM, 36.01 ± 5.53 ng/mg tissue weight (t.w.)) treatment compared to control (264.56 ± 15.46 ng/mg t.w.). R-PIA inhibition (0.01 µM = 141.97 ± 15.79 vs control = 244.77 ± 13.79 ng/mg t.w.) of PRL release was blocked by 1 µM cyclopentyltheophylline, a specific A1 receptor antagonist (1 µM = 212.360 ± 26.560 ng/mg t.w.), whereas cyclopentyltheophylline alone (0.01, 0.1, 1 µM) had no effect. R-PIA (0.001, 0.01, 0.1, 1 µM) produced inhibition of PRL secretion stimulated by both phospholipase C (0.5 IU/mL; 977.44 ± 76.17 ng/mg t.w.) and dibutyryl cAMP (1 mM; 415.93 ± 37.66 ng/mg t.w.) with nadir established at the dose of 0.1 µM (225.55 ± 71.42 and 201.9 ± 19.08 ng/mg t.w., respectively). Similarly, R-PIA (0.01 µM) decreased (242.00 ± 24.00 ng/mg t.w.) the PRL secretion stimulated by cholera toxin (0.5 mg/mL; 1050.00 ± 70.00 ng/mg t.w.). In contrast, R-PIA had no effect (468.00 ± 34.00 ng/mg t.w.) on PRL secretion stimulation by pertussis toxin (0.5 mg/mL; 430.00 ± 26.00 ng/mg t.w.). These results suggest that inhibition of PRL secretion after A1 receptor activation by R-PIA is mediated by a Gi protein-dependent mechanism.