Angiotensin II involvement in the development and persistence of amphetamine-induced sensitization: Striatal dopamine reuptake implications.

Amphetamine (AMPH) exposure induces behavioural and neurochemical sensitization observed in rodents as hyperlocomotion and increased dopamine release in response to a subsequent dose. Brain Angiotensin II modulates dopaminergic neurotransmission through its AT1 receptors (AT1-R), positively regulating striatal dopamine synthesis and release. This work aims to evaluate the AT1-R role in the development and maintenance of AMPH-induced sensitization. Also, the AT1-R involvement in striatal dopamine reuptake was analysed. The sensitization protocol consisted of daily AMPH administration for 5 days and tested 21 days after withdrawal. An AT1-R antagonist, candesartan, was administered before or after AMPH exposure to evaluate the participation of AT1-R in the development and maintenance of sensitization, respectively. Sensitization was evaluated by locomotor activity and c-Fos immunostaining. Changes in dopamine reuptake kinetics were evaluated 1 day after AT1-R blockade withdrawal treatment, with or without the addition of AMPH in vitro. The social interaction test was performed as another behavioural output. Repeated AMPH exposure induced behavioural and neurochemical sensitization, which was prevented and reversed by candesartan. The AT1-R blockade increased the dopamine reuptake kinetics. Neither the AMPH administration nor the AT1-R blockade altered the performance of social interaction. Our results highlight the AT1-R's crucial role in AMPH sensitization. The enhancement of dopamine reuptake kinetics induced by the AT1-R blockade might attenuate the neuroadaptive changes that lead to AMPH sensitization and its self-perpetuation. Therefore, AT1-R is a prominent candidate as a target for pharmacological treatment of pathologies related to dopamine imbalance, including drug addiction and schizophrenia.

Opioid modulation of prolactin secretion induced by stress during late pregnancy. Role of ovarian steroids.

BACKGROUND: The opioid system modulates prolactin release during late pregnancy. Its role and the participation of ovarian hormones in this modulation are explored in ether stress-induced prolactin release. METHODS/RESULTS: Estrous, 3-day and 19-day pregnant rats were used. We administered the antagonist mifepristone (Mp) and tamoxifen to evaluate progesterone and estradiol action in naloxone (NAL, opioid antagonist) or saline treated rats. Ether stress had no effect on serum prolactin levels in controls but increased prolactin release in NAL-treated rats. Prolactin response to stress in NAL-treated rats was blocked by l-DOPA administration. Mp treatment on day 18 of pregnancy increased prolactin levels after stress without alterations by NAL. Tamoxifen on days 14 and 15 of pregnancy completely blocked Mp and NAL effects on prolactin release at late pregnancy. In contrast, stress significantly increased prolactin levels in estrous rats and pretreatment with NAL prevented this. On day 3 of pregnancy, at 6.00 p.m., stress and NAL treatment inhibited prolactin levels in saline-treated rat. No effect of stress or NAL administration was detected on day 3 of pregnancy at 9.00 a.m. icv administration of specific opioids antagonist, B-Funaltrexamine but not Nor-Binaltorphimine or Naltrindole, caused a significant increase in stress-induced prolactin release. CONCLUSIONS: Opioid system suppression of prolactin stress response during late pregnancy was observed only after progesterone withdrawal, involving a different opioid mechanism from its well-established stimulatory role. This mechanism acts through a mu opioid receptor and requires estrogen participation. The opioid system and progesterone may modulate stress-induced prolactin release, probably involving a putative prolactin-releasing factor.

Angiotensin II AT1 receptors are involved in neuronal activation induced by amphetamine in a two-injection protocol.

It was already found that Ang II AT1 receptors are involved in the neuroadaptative changes induced by a single exposure to amphetamine, and such changes are related to the development of behavioral and neurochemical sensitization. The induction of the immediately early gene c-fos has been used to define brain activated areas by amphetamine. Our aim was to evaluate the participation of AT1 receptors in the neuronal activation induced by amphetamine sensitization. The study examined the c-fos expression in mesocorticolimbic areas induced by amphetamine challenge (0.5 mg/kg i.p) in animals pretreated with candesartan, a selective AT1 receptor blocker (3 mg/kg p.o × 5 days), and amphetamine (5 mg/kg i.p) 3 weeks before the challenge. Increased c-fos immunoreactivity was found in response to the amphetamine challenge in the dorsomedial caudate-putamen and nucleus accumbens, and both responses were blunted by the AT1 receptor blocker pretreatment. In the infralimbic prefrontal cortex, increased c-fos immunoreactivity was found in response to amphetamine and saline challenge, and both were prevented by the AT1 receptor blocker. No differences were found neither in ventral tegmental area nor prelimbic cortex between groups. Our results indicate an important role for brain Ang II in the behavioral and neuronal sensitization induced by amphetamine.

Alpha and beta noradrenergic mediation of NMDA glutamatergic effects on lordosis behaviour and plasmatic LH concentrations in the primed female rat.

In previous studies we have found that blockade of NMDA (N-Methyl-D-Aspartic-Acid)-type glutamatergic receptor with intracerebroventricular (ICV) selective drugs induces an inhibition of lordosis in ovariectomized (OVX) estrogen primed rats receiving progesterone or luteinizing hormone releasing hormone (LHRH). By the opposite way, stimulation with NMDA in OVX estrogen primed rats induced a significant increase of lordosis. In the present study the action of an alpha1-noradrenergic antagonist, HEAT (BE 2254/2-beta-4-Hydroxyphenyl-Ethyl-Aminomethyl-1-Tetralone), and Metoprolol, a beta-noradrenergic antagonist, were studied injecting them ICV previously to NMDA administration in treated OVX estrogen primed rats. In experiment 1, the enhancing effect on lordosis induced by NMDA at high dose (1 microg) was abolished by HEAT administration (P < 0.001 for 3 and 6 microg), and the LH plasma levels were decreased only with the higher dose (P < 0.05), suggesting that behavioral effects are quite more sensitive to the alpha-blockade than hormonal effects. In experiment 2, enhancing effects on lordosis behavior were not observed with neither the NMDA at low dose (0.5 microg) nor the metoprolol alone (5.71 microg), but a synergism was observed when both were simultaneously administered (P < 0.001). The LH plasma levels were increased by Metoprolol alone (P < 0.05), and powered by the combination with NMDA at low dose (P < 0.01 vs. SAL and NMDA alone); no differences were observed with Metoprolol. LH increase was observed with Metoprolol even without behavioural modifications. These findings strongly suggest that facilitatory and inhibitory effects of NMDA in this model are mediated by alpha- and beta-adrenergic transmission in both, behavioral and hormonal effects.

A centrally acting, anxiolytic angiotensin II AT1 receptor antagonist prevents the isolation stress-induced decrease in cortical CRF1 receptor and benzodiazepine binding.

Long-term pretreatment with an angiotensin II AT1 antagonist blocks angiotensin II effects in brain and peripheral organs and abolishes the sympathoadrenal and hypothalamic-pituitary-adrenal responses to isolation stress. We determined whether AT1 receptors were also important for the stress response of higher regulatory centers. We studied angiotensin II and corticotropin-releasing factor (CRF) receptors and benzodiazepine binding sites in brains of Wistar Hannover rats. Animals were pretreated for 13 days with vehicle or a central and peripheral AT1 antagonist (candesartan, 0.5 mg/kg/day) via osmotic minipumps followed by 24 h of isolation in metabolic cages, or kept grouped throughout the study (grouped controls). In another study, we determined the influence of a similar treatment with candesartan on performance in an elevated plus-maze. AT1 receptor blockade prevented the isolation-induced increase in brain AT1 receptors and decrease in AT2 binding in the locus coeruleus. AT1 receptor antagonism also prevented the increase in tyrosine hydroxylase mRNA in the locus coeruleus. Pretreatment with the AT1 receptor antagonist completely prevented the decrease in cortical CRF1 receptor and benzodiazepine binding produced by isolation stress. In addition, pretreatment with candesartan increased the time spent in and the number of entries to open arms of the elevated plus-maze, measure of decreased anxiety. Our results implicate a modulation of upstream neurotransmission processes regulating cortical CRF1 receptors and the GABA(A) complex as molecular mechanisms responsible for the anti-anxiety effect of centrally acting AT1 receptor antagonists. We propose that AT1 receptor antagonists can be considered as compounds with possible therapeutic anti-stress and anti-anxiety properties.

Matrix metalloproteinase-2 cleavage of adrenomedullin produces a vasoconstrictor out of a vasodilator.

MMPs (matrix metalloproteinases) play a major role in the pathogenesis of hypertension by altering the extracellular matrix during cardiovascular remodelling. In the present study we show that MMP-2, but not MMP-9, cleaves the vasodilator peptide AM (adrenomedullin). Addition of the AM-binding protein, complement factor H, prevents this cleavage, providing a hitherto unknown mechanism of action for this binding protein. We identified the signature cleavage fragments and found some of them in human urine, suggesting that MMP-2 processing of AM may occur in vivo. Synthetic AM fragments regulated blood pressure in rats. The larger peptides are vasodilators, as is intact AM, whereas intermediate fragments did not affect blood pressure. In contrast, AM(11-22) elicited vasoconstriction. Studies of AM receptor activation in Rat2 cells confirm that the larger AM cleavage peptides activated this receptor, whereas AM(11-22) did not. The present study defines a new mechanism through which MMP-2 may regulate blood pressure by simultaneously eliminating a vasodilator and generating a vasoconstrictor.

NMDA receptors in the medial zona incerta stimulate luteinizing hormone and prolactin release.

1. The aim of the present work is to demonstrate the interaction between the glutamatergic/NMDA and dopaminergic systems in the medial zona incerta on the control of luteinizing hormone and prolactin secretion and the influence of reproductive hormones. 2. Proestrus and ovariectomized rats were primed with estrogen and progesterone to induce high or low levels of luteinizing hormone and prolactin. 2-Amino-7-phosphonoheptanoic acid, an NMDA receptor antagonist, and dopamine were injected in the medial zona incerta. Blood samples were withdrawn every hour between 1,600 and 2,000 hours or 2,200 hours via intracardiac catheter from conscious rats. Additional groups of animals injected with the NMDA receptor antagonist were killed 1 or 4 h after injection. Dopamine and its metabolite 3,4-dihydroxyphenylacetic acid were measured in different hypothalamic regions. 3. 2-Amino-7-phosphonoheptanoic acid blocked the ovulatory luteinizing hormone surge in proestrus rats. 2-Amino-7-phosphonoheptanoic acid also blocked the increase in luteinizing hormone induced by ovarian hormones in ovariectomized rats, an effect that was partially reversed by dopamine injection. Conversely, the increased release of luteinizing hormone and prolactin induced by dopamine was prevented by 2-amino-7-phosphonoheptanoic acid. We found that the NMDA antagonist injection decreased the dopaminergic activity--as evaluated by the 3,4-dihydroxyphenylacetic acid/dopamine ratio--in the medio basal hypothalamus and increased in the preoptic area. 4. Our results show an stimulatory role of NMDA receptors on the ovulatory luteinizing hormone release and on luteinizing hormone release induced by sexual hormones and demonstrate that the stimulatory effect of dopamine on luteinizing hormone and prolactin is mediated by the NMDA receptors. These results suggest a close interaction between the glutamatergic and dopaminergic incertohypothalamic systems on the control of luteinizing hormone and prolactin release.

Identification of vasoactive nonpeptidic positive and negative modulators of adrenomedullin using a neutralizing antibody-based screening strategy.

Adrenomedullin (AM) is a peptide hormone implicated in blood pressure regulation and in the pathophysiology of important diseases, such as hypertension, cancer, and diabetes. However, nonpeptidic modulators of this peptide that could be used to clinically regulate its actions are not available. We present here an efficient new method to screen a large library of small molecules. This technology was applied to the identification of positive and negative modulators of AM function. A two-tier screening strategy was developed in which the first screening entails disruption of the interaction between the peptide and a neutralizing monoclonal antibody. Selected compounds were further characterized by their ability to modulate second messengers in cells containing specific AM receptors. A parallel screen against gastrin-releasing peptide selected a different subset of molecules, confirming the specificity of the screening method. Identified AM-positive regulators reduced blood pressure in vivo, whereas AM-negative regulators mediated vasoconstriction, as predicted by the vasodilatory activity of AM. Binding of the small molecules to immobilized AM was demonstrated by surface plasmon resonance assays, with K(d) values ranging from 7.76 x 10(-9) to 4.14 x 10(-6) m. Preclinical development of AM modulators may result in useful drugs for the prevention and treatment of hypertension, cancer, and diabetes.

Anti-inflammatory effects of angiotensin II AT1 receptor antagonism prevent stress-induced gastric injury.

Stress reduces gastric blood flow and produces acute gastric mucosal lesions. We studied the role of angiotensin II in gastric blood flow and gastric ulceration during stress. Spontaneously hypertensive rats were pretreated for 14 days with the AT1 receptor antagonist candesartan before cold-restraint stress. AT1 receptors were localized in the endothelium of arteries in the gastric mucosa and in all gastric layers. AT1 blockade increased gastric blood flow by 40-50%, prevented gastric ulcer formation by 70-80% after cold-restraint stress, reduced the increase in adrenomedullary epinephrine and tyrosine hydroxylase mRNA without preventing the stress-induced increase in adrenal corticosterone, decreased the stress-induced expression of TNF-alpha and that of the adhesion protein ICAM-1 in arterial endothelium, decreased the neutrophil infiltration in the gastric mucosa, and decreased the gastric content of PGE2. AT1 receptor blockers prevent stress-induced ulcerations by a combination of gastric blood flow protection, decreased sympathoadrenal activation, and anti-inflammatory effects (with reduction in TNF-alpha and ICAM-1 expression leading to reduced neutrophil infiltration) while maintaining the protective glucocorticoid effects and PGE2 release. Angiotensin II has a crucial role, through stimulation of AT1 receptors, in the production and progression of stress-induced gastric injury, and AT1 receptor antagonists could be of therapeutic benefit.

Allopregnanolone increase in striatal N-methyl-D-aspartic acid evoked [3H]dopamine release is estrogen and progesterone dependent.

1. The neurosteroids are compounds derived from steroid hormones and synthesized in the nervous system. They can modulate different neurotransmitter pathways. In previous work we demonstrated that progesterone modulates dopamine release induced by the glutamatergic agonist N-methyl-D-aspartic acid (NMDA). 2. The aim of this work was to evaluate a possible modulatory role of the progesterone metabolite allopregnanolone on NMDA-evoked [3H]dopamine release from corpus striatum slices obtained from cycling and ovariectomized female rats. 3. We used a dynamic superfusion method to evaluate the release of [3H]dopamine. Allopregnanolone at 50-600 nM was added to the superfusion buffer (Krebs-Ringer-bicarbonate-glucose. pH 7.4. with constant O2/CO2 gassing). The results are expressed as a percentage over basal [3H]dopamine loaded by the tissue. 4. Allopregnanolone (50 and 100 nM) increased the NMDA-evoked [3H]dopamine release from estrus rats. The remaining doses did not show significant changes in the pattern of release. This effect was not observed in diestrus rats. The ovariectomy abolished the facilitatory effect of allopregnanolone on NMDA-evoked 2 [3H]dopamine release. 5. Subcutaneous administration of exogenous estrogen (25 mg/rat) and progesterone (1 mg/rat) restored the facilitatory effect on dopaminergic input. 6. These results suggest that allopregnanolone is a neurosteroid able to modulate dopamine release in an ovarian-hormone-fluctuation-dependent manner and provide further support for a role of allopregnanolone as a modulator of glutamatergic-dopaminergic interaction in the corpus striatum.