1A Antagonist Research Articles

Adenosine Receptor Stimulation Improves Glucocorticoid-Induced Osteoporosis in a Rat Model.

Glucocorticoid-induced osteoporosis (GIO) is a secondary cause of bone loss. Bisphosphonates approved for GIO, might induce jaw osteonecrosis; thus additional therapeutics are required. Adenosine receptor agonists are positive regulators of bone remodeling, thus the efficacy of adenosine receptor stimulation for treating GIO was tested. In a preventive study GIO was induced in Sprague-Dawley rats by methylprednisolone (MP) for 60 days. Animals were randomly assigned to receive polydeoxyribonucleotide (PDRN), an adenosine A2 receptor agonist, or PDRN and DMPX (3,7-dimethyl-1-propargylxanthine, an A2 antagonist), or vehicle (0.9% NaCl). Another set of animals was used for a treatment study, following the 60 days of MP-induction rats were randomized to receive (for additional 60 days) PDRN, or PDRN and DMPX (an adenosine A2 receptor antagonist), or zoledronate (as control for gold standard treatment), or vehicle. Control animals were administered with vehicle for either 60 or 120 days. Femurs were analyzed after treatments for histology, imaging, and breaking strength analysis. MP treatment induced severe bone loss, the concomitant use of PDRN prevented the developing of osteoporosis. In rats treated for 120 days, PDRN restored bone architecture and bone strength; increased b-ALP, osteocalcin, osteoprotegerin and stimulated the Wnt canonical and non-canonical pathway. Zoledronate reduced bone resorption and ameliorated the histological features, without significant effects on bone formation. Our results suggest that adenosine receptor stimulation might be useful for preventing and treating GIO.

5-Substituted 2-benzylidene-1-tetralone analogues as A1 and/or A2A antagonists for the potential treatment of neurological conditions

Adenosine A1 and A2A receptors are attracting great interest as drug targets for their role in cognitive and motor deficits, respectively. Antagonism of both these adenosine receptors may offer therapeutic benefits in complex neurological diseases, such as Alzheimer’s and Parkinson’s disease. The aim of this study was to explore the affinity and selectivity of 2-benzylidene-1-tetralone derivatives as adenosine A1 and A2A receptor antagonists. Several 5-hydroxy substituted 2-benzylidene-1-tetralone analogues with substituents on ring B were synthesized and assessed as antagonists of the adenosine A1 and A2A receptors via radioligand binding assays. The results indicated that hydroxy substitution in the meta and para position of phenyl ring B, displayed the highest selectivity and affinity for the adenosine A1 receptor with Ki values in the low micromolar range. Replacement of ring B with a 2-amino-pyrimidine moiety led to compound 12 with an increase of affinity and selectivity for the adenosine A2A receptor. These substitution patterns led to enhanced adenosine A1 and A2A receptor binding affinity. The para-substituted 5-hydroxy analogue 3 behaved as an adenosine A1 receptor antagonists in a GTP shift assay performed with rat whole brain membranes expressing adenosine A1 receptors. In conclusion, compounds 3 and 12, showed the best adenosine A1 and A2A receptor affinity respectively, and therefore represent novel adenosine receptor antagonists that may have potential with further structural modifications as drug candidates for neurological disorders.

Synergic stimulation of serotonin 5-HT1A receptor and α2-adrenoceptors for neuropathic pain relief: Preclinical effects of 2-substituted imidazoline derivatives

Neuropathic pain affects millions of people causing disability and impairing quality of life. Commonly used analgesics are generally characterized by limited therapeutic outcomes. The serotonin 5‐HT1A receptor and the α2 adrenergic receptors are involved in central nociceptive mechanisms with a pivotal role in the inhibitory descending pain pathway. Since their stimulation may modulate the nervous signaling altered by neuropathies, the purpose of the present research is the study of the combined activation of 5‐HT1A and α2 receptors by rationally designed imidazoline ligands ((S)-(-)-1 and 2–5) in a rat model of neuropathic pain (chronic constriction injury - CCI). On day 14 after nerve damage, the acute administration per os (p.o.) of low doses of (S)-(-)-1 (0.1–1mg/kg) was able to significantly increase the pain threshold to mechanical noxious stimuli for more than 1h. (S)-(-)-1 efficacy was confirmed by the decrease of spontaneous pain evaluated as hind limb weight bearing alterations. The clinically-used compound gabapentin (100mg/kg p.o.) induced a pain relieving effect similar to (S)-(-)-1 administered at 100 fold lower dose. In the same model, the selected analogues, compounds 2–5 (1mg/kg p.o.) were effective 30min after administration. In particular, 5 fully reverted the CCI-induced hypersensitivity. The pain relieving activity of 5 was significantly prevented by the selective 5-HT1A receptor antagonist WAY 100635 (1mg/kg intraperitoneally, i.p.) and, at a lesser extent, by the α2 antagonist yohimbine (3mg/kg i.p.). A novel pharmacodynamic approach to the treatment of neuropathic pain is presented.

Alpha-bisabolol Promotes Glioma Cell Death by Modulating the Adenosinergic System.

Glioblastoma multiforme is the most malignant type of glioma. Alpha-bisabolol is an essential oil reported as a potent cell death agent. In the present work, we evaluated the effect of alpha-bisabolol on ecto-5'-nucleotidase/CD73, the most well-characterized enzymatic source of adenosine, present in lipid rafts. Glioma cells were treated with alpha-bisabolol and, in some experiments, pre-treated with an A3 antagonist. MTT assay (viability), malachite green method (ecto-5'-nucleotidase/CD73 activity) and quantitative polymerase chain reaction (qPCR) (A3 mRNA) were carried out. Alpha-bisabolol led to a decrease in C6 and U138-MG glioma cells viability, accompanied by an increase in ecto-5'-NT/CD73 activity. Pre-treatment with an A3 antagonist reverted the effect of α-bisabolol with an increase of mRNA expression of this receptor. Our data indicated the participation of ecto-5'-nucleotidase/CD73 and A3 receptor in the anti-proliferative effect of α-bisabolol on glioma cells.

Role of Alpha‐adrenergic Inputs to PVN in the Cardiovascular Responses to Acute Hypoxia

Previous studies indicate that catecholaminergic neurons in nucleus of solitary tract (NTS) contribute to intermittent hypoxia hypertension and that the hypothalamic paraventricular nucleus (PVN) plays a role in the cardiovascular response to hypoxia. The present study was designed to examine the role of alpha‐adrenergic receptors in PVN in the cardiovascular responses to acute hypoxia. Rats were anesthetized with chloralose and urethane and mean arterial pressure (MAP), heart rate (HR), renal sympathetic nerve activity (RSNA) and end tidal CO2 measured. The rat was placed in a stereotaxic frame for microinjections in PVN. After paralysis with gallamine, the rat was artificially ventilated with room air. Hypoxia was induced by ventilating the animal with 10% O2 for 30 seconds. When the parameters returned to the baseline, bilateral injections of vehicle or either α1 antagonist prazosin (500nmol) or α2 antagonist idazoxan (5nmol) were made in PVN and responses to hypoxia observed after 20 minutes. At the end of experiment brain was removed for localization of microinjection site. Hypoxia increased MAP with no change in HR or RSNA. The increase in MAP induced by systemic hypoxia was significantly blunted by bilateral administration of either prazosin (36 ± 3mmHg to 19 ± 4mmHg, n=11) or idazoxan (36 ± 2mmHg to 25 ± 5mmHg, n=10) in PVN. Changes in baseline blood pressure were not significant following prazosin (−2 ± 4mmHg) or idazoxan (0 ± 3mmHg). The results suggest that catecholaminergic inputs to PVN play a role in acute hypoxia induced increases in MAP.Support or Funding InformationSupported by PO1 HL88052

Retinal A2A and A3 adenosine receptors modulate the components of the rat electroretinogram.

Adenosine is a neuromodulator present in various areas of the central nervous system, including the retina. Adenosine may serve a neuroprotective role in the retina, based on electroretinogram (ERG) recordings from the rat retina. Our purpose was to assess the role of A2A and A3 adenosine receptors in the generation and modulation of the rat ERG. The flash ERG was recorded with corneal electrodes from Sprague Dawley rats. Agonists and antagonists for A2A and A3 receptors, and adenosine were injected (5 µl) into the vitreous. The effects on the components of the single flash scotopic and photopic ERGs were examined, and ERG flicker. Adenosine (0.5 mM) increased the mean amplitudes of the scotopic ERG a-waves (68 ± 8 to 97 ± 14 µV, P = 0.042), and b-waves (236 ± 38 µV to 305 ± 42 µV). A2A agonist CGS21680 (2 mM) reduced the mean amplitude of the ERG b-wave, from 298 ± 21 µV in response to the brightest stimulus to 212 ± 19 µV (P = 0.005), and mean scotopic oscillatory potentials (OPs) from 100 ± 9 µV to 47 ± 11 µV (P = 0.023). ZM241385 [4 mM], an A2A antagonist, decreased the scotopic b-wave of the ERG. A3 agonist 2-CI-IB-MECA (0.5 mM) increased the a-wave, while decreasing the scotopic and photopic ERG b-waves, and the scotopic OPs. A3 antagonist VUF5574 (1 mM) increased the mean amplitude of the scotopic a-wave (66 ± 8 to 140 ± 29 µV, P = 0.046) and b-wave (224 ± 20 to 312 ± 39 µV, P = 0.0037). No significant effects on ERG flicker were found. We conclude that retinal neurons containing A2A and/or A3 adenosine receptors contribute to the generation of the ERG a- and b-waves and OPs.

Correlation of transient adenosine release and oxygen changes in the caudate-putamen.

Adenosine is an endogenous nucleoside that modulates important physiological processes, such as vasodilation, in the central nervous system. A rapid, 2-4s, mode of adenosine signaling has been recently discovered, but the relationship between this type of adenosine and blood flow change has not been characterized. In this study, adenosine and oxygen changes were simultaneously measured using fast-scan cyclic voltammetry. Oxygen changes occur when there is an increase in local cerebral blood flow and thus are a measure of vasodilation. About 34% of adenosine transients in the rat caudate-putamen are correlated with a subsequent transient change in oxygen. The amount of oxygen was correlated with the concentration of adenosine release and larger adenosine transients (over 0.4μM) always had subsequent oxygen changes. The average duration of adenosine and oxygen transients was 3.2 and 3.5s, respectively. On average, the adenosine release starts and peaks 0.2s prior to the oxygen. The A2a antagonist, SCH442416, decreased the number of both adenosine and oxygen transient events by about 32%. However, the A1 antagonist, DPCPX, did not significantly affect simultaneous adenosine and oxygen release. The nitric oxide (NO) synthase inhibitor l-NAME also did not affect the concentration or number of adenosine and oxygen release events. These results demonstrate that both adenosine and oxygen release are modulated via A2a receptors. The correlation of transient concentrations, time delay between adenosine and oxygen peaks, and effect of A2a receptors suggests that adenosine modulates blood flow on a rapid, sub-second time scale. Read the Editorial Highlight for this article on page 10.

Pulmonary platelet accumulation induced by catecholamines: Its involvement in lipopolysaccharide-induced anaphylaxis-like shock

Intravenously injected lipopolysaccharides (LPS) rapidly induce pulmonary platelet accumulation (PPA) and anaphylaxis-like shock (ALS) in mice. Macrophages reportedly release catecholamines rapidly upon stimulation with LPS. Here, we examined the involvement of macrophage-derived catecholamines in LPS-induced PPA and ALS. A catecholamine or Klebsiella O3 (KO3) LPS was intravenously injected into mice, with 5-hydroxytryptamine in the lung being measured as a platelet marker. The tested catecholamines induced PPA, leading to shock. Their minimum shock-inducing doses were at the nmol/kg level. The effects of epinephrine and norepinephrine were inhibited by prazosin (α1 antagonist) and by yohimbine (α2 antagonist), while dopamine's were inhibited only by prazosin. Use of synthetic adrenergic α1- and/or α2-agonists, platelet- or macrophage-depleted mice, a complement C5 inhibitor and C5-deficient mice revealed that (a) α2-receptor-mediated PPA and shock depend on both macrophages and complements, while α1-receptor-mediated PPA and shock depend on neither macrophages nor complements, (b) the PPA and ALS induced by KO3-LPS depend on α1- and α2-receptors, macrophages, and complements, and (c) KO3-LPS-induced PPA is preceded by catecholamines decreasing in serum. Together, these results suggest the following. (i) Catecholamines may stimulate macrophages and release complement C5 via α2-receptors. (ii) Macrophage-derived catecholamines may mediate LPS-induced PPA and ALS. (iii) Moderate PPA may serve as a defense mechanism to remove excess catecholamines from the circulation by promoting their rapid uptake, thus preventing excessive systemic effects. (iv) The present findings might provide an insight into possible future pharmacological strategies against such diseases as shock and acute respiratory distress syndrome.

Ethanol and Caffeine Effects on Social Interaction and Recognition in Mice: Involvement of Adenosine A2A and A1 Receptors.

Ethanol and caffeine are frequently consumed in combination and have opposite effects on the adenosine system: ethanol metabolism leads to an increase in adenosine levels, while caffeine is a non-selective adenosine A1/A2A receptor antagonist. These receptors are highly expressed in striatum and olfactory tubercle, brain areas involved in exploration and social interaction in rodents. Ethanol modulates social interaction processes, but the role of adenosine in social behavior is still poorly understood. The present work was undertaken to study the impact of ethanol, caffeine and their combination on social behavior, and to explore the involvement of A1 and A2A receptors on those actions. Male CD1 mice were evaluated in a social interaction three-chamber paradigm, for preference of conspecific vs. object, and also for long-term recognition memory of familiar vs. novel conspecific. Ethanol showed a biphasic effect, with low doses (0.25 g/kg) increasing social contact and higher doses (1.0–1.5 g/kg) reducing social interaction. However, no dose changed social preference; mice always spent more time sniffing the conspecific than the object, independently of the ethanol dose. Ethanol, even at doses that did not change social exploration, produced amnestic effects on social recognition the following day. Caffeine reduced social contact (15.0–60.0 mg/kg), and even blocked social preference at higher doses (30.0–60.0 mg/kg). The A1 antagonist Cyclopentyltheophylline (CPT; 3–9 mg/kg) did not modify social contact or preference on its own, and the A2A antagonist MSX-3 (1.5–6 mg/kg) increased social interaction at all doses. Ethanol at intermediate doses (0.5–1.0 g/kg) was able to reverse the reduction in social exploration induced by caffeine (15.0–30.0 mg/kg). Although there was no interaction between ethanol and CPT or MSX-3 on social exploration in the first day, MSX-3 blocked the amnestic effects of ethanol observed on the following day. Thus, ethanol impairs the formation of social memories, and A2A adenosine antagonists can prevent the amnestic effects of ethanol, so that animals can recognize familiar conspecifics. On the other hand, ethanol can counteract the social withdrawal induced by caffeine, a non-selective adenosine A1/A2A receptor antagonist. These results show the complex set of interactions between ethanol and caffeine, some of which could be the result of the opposing effects they have in modulating the adenosine system.

A Single Dose, Randomized, Controlled Proof-Of-Mechanism Study of a Novel Vasopressin 1a Receptor Antagonist (RG7713) in High-Functioning Adults with Autism Spectrum Disorder

The core symptoms of autism spectrum disorder (ASD) include impaired social communication, repetitive behaviors, and restricted interests. No effective pharmacotherapy for these core deficits exists. Within the domain of social communication, the vasopressin system is implicated in social cognition and social signaling deficits of ASD, and represents a potential therapeutic target. We assessed the effects of a single 20 mg intravenous dose of the arginine vasopressin receptor 1A (V1a) antagonist, RG7713, on exploratory biomarkers (eye tracking), behavioral and clinical measures of social cognition and communication (affective speech recognition (ASR), reading the mind in the eyes, olfactory identification, scripted interaction), and safety and tolerability in a multicenter, randomized, double-blind, placebo-controlled, cross-over study of 19 high-functioning adult male subjects with DSM-IV Autistic Disorder (age 18–45 years; full scale IQ >70; ABC-Irritability subscale ⩽13). Eye-tracking showed an increase in biological motion orienting preference with RG7713 (ES=0.8, p=0.047) and a non-significant improvement in the composite score (ES=0.2, p=0.29). RG7713 reduced ability to detect lust (ES=−0.8, p=0.03) and fear (ES=−0.7, p=0.07) in ASR. However, when all eight individual emotion subscales were combined into an overall ASR performance score, the reduction was non-significant (ES=−0.1, p=0.59). Thirteen adverse events were reported in 10 subjects; all were of mild (11/13) or moderate (2/13) severity. Although interpretation should be cautious due to multiple comparisons and small sample size, these results provide preliminary evidence from experimental and behavioral biomarkers, that blockade of the V1a receptor may improve social communication in adults with high-functioning ASD. ClinicalTrials.gov identifier: NCT01474278 A Study of RO5028442 in Adult Male High-Functioning Autistic Patients. Available at: https://clinicaltrials.gov/ct2/show/NCT01474278

Activation of arginine vasopressin receptor 1a facilitates the induction of long-term potentiation in the accessory olfactory bulb of male mice

Olfaction plays an important role in social recognition in most mammals. Central arginine vasopressin (AVP) plays a role in this olfaction-based recognition. The high level of expression of AVP receptors in the accessory olfactory bulb (AOB) at the first relay of the vomeronasal system highlights the importance of AVP signaling at this stage. We therefore analyzed the effects of AVP on the synaptic plasticity of glutamatergic transmission from mitral cells to granule cells in AOB slices from male mice. To monitor the strength of the glutamatergic transmission, we measured the maximal initial slope of the lateral olfactory tract-evoked field potential, which represents the granule cell response to mitral cell activation. AVP paired with 100-Hz stimulation that only produced short-term potentiation enhanced the induction of long-term potentiation (LTP) in a dose-dependent manner. AVP-paired LTP was blocked by the selective AVP receptor 1a (AVPR1a) antagonist, d(CH2)5[Tyr(Me)2]AVP (Manning compound), but not by the AVPR1b antagonist SSR149415, and it was mimicked by the selective AVPR1a agonist [Phe2, Ile3, Orn8]-vasopressin. We further examined the effect of AVP on the reciprocal transmission between mitral and granule cells by stimulating a mitral cell and recording the evoked inhibitory postsynaptic currents (IPSCs) from the same cell using conventional whole-cell patch-clamp techniques. AVP reduced the reciprocal IPSCs triggered by endogenous glutamate release from the excited mitral cell. These results suggest that AVP promotes the induction of LTP at the mitral-to-granule cell synapse via the activation of AVPR1a through an as-yet-to-be-determined mechanism in the AOB of male mice.

Pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidines and Structurally Simplified Analogs. Chemistry and SAR Profile as Adenosine Receptor Antagonists.

Adenosine was defined as a neuromodulator which exerts its action by interaction with specific G-protein coupled receptor termed adenosine receptors. Adenosine receptors are expressed in several tissues and cells of our body and exist as four different subtypes of these receptors: A1, A2, A2B and A3. In the last years significant efforts were made to obtain highly potent and selective ligands for the four adenosine receptors subtypes. Both agonists and antagonists were used as pharmacological tools to study therapeutic implications of enhancing or blocking the adenosine receptors activity, and some of these compounds have reached clinical phases. The pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidines (PTPs) represent one of the several templates designed as adenosine receptor antagonists. A lot of synthetic work was made on this scaffold in order to obtain potent A2 and A3 antagonists. Here were reviewed the synthetic approaches followed by both academia and industry to introduce different substituents at different positions of the PTP nucleus, in particular at the 2, 5, 7, 8 and 9 positions. Nevertheless PTP derivatives are tricyclic compounds with a high molecular weight which exhibit limitations such as poor aqueous solubility and difficult synthetic preparation. With the aim to obtain derivatives with the same potency and selectivity of PTP but with better drug-like properties, researchers made structural simplification of this scaffold. Replacement of the pyrazole or triazole rings of PTP led to the [1,2,4]triazolo[1,5-c]pyrimidine and pyrazolo[3,4- d]pyrimidine derivatives, respectively. Synthetic strategies for these compounds were reported, combined with the SAR profile on the adenosine receptors.

From Bench to Bedside and Back Again: A Personal Journey with Dexmedetomidine.

Dexmedetomidine Diminishes Halothane Anesthetic Requirements in Rats Through a Postsynaptic Alpha 2 Adrenergic Receptor. By Segal IS, Vickery RG, Walton JK, Doze VA, and Maze M. ANESTHESIOLOGY 1988; 125:590-4. Abstract reprinted with permission.The effect of 4(5)-[1-(2,3-dimethylphenyl)ethyl]imidazole (medetomidine), the α2 adrenergic agonist, on anesthetic requirements was investigated in rats anesthetized with halothane. Halothane MAC was determined before and after either dexmedetomidine (D-enantiomer) or levomedetomidine (L-enantiomer) 10, 30, and 100 μg/kg, or vehicle intraperitoneally. There was a dose-dependent increase in MAC with the D-, but not the L-, stereoisomer. At the highest dose of dexmedetomidine (100 μg/kg), halothane could be discontinued for up to 30 min with no response to tail clamping. To determine whether α2 adrenoreceptors mediated this effect of dexmedetomidine on MAC, cohorts of rats were pretreated with idazoxan, 10 mg/kg intraperitoneally, a highly selective α2 antagonist. This completely prevented the reduction of MAC caused by dexmedetomidine. To determine whether the reduction of MAC caused by dexmedetomidine was mediated in part through either opiate or adenosine receptors, groups of rats were pretreated with either naltrexone, 5 mg/kg intraperitoneally, an opiate antagonist, or 8-phenyltheophylline, 2.5 mg/kg intraperitoneally, an A1 adenosine antagonist. These two pretreatments did not alter the reduction of MAC by dexmedetomidine. To determine whether postsynaptic mechanisms mediate the anesthetic effect of dexmedetomidine, rats were depleted of central catecholamine stores with either n-(2-chloroethyl)-n-ethyl-2-bromobenzylamine or reserpine and α-methyl-para-tyrosine, and MAC was determined before and after each dose of dexmedetomidine. While the catecholamine-depleted rats had a lower basal MAC than the vehicle controls, there was still a profound reduction in halothane MAC after administration of dexmedetomidine. The reduction of MAC by dexmedetomidine was blocked with idazoxan in the catecholamine-depleted rats. These data indicate that the reduction of MAC caused by dexmedetomidine is mediated through α2 adrenoreceptors with no apparent involvement of either opiate or A1 adenosine receptors. Data from catecholamine-depleted rats suggest that the mediating mechanism must involve site(s) other than or in addition to the presynaptic α2 adrenergic receptors on noradrenergic neurons. The authors conclude that central postsynaptic α2 adrenergic receptors mediate a significant part of the reduction of anesthetic requirements caused by dexmedetomidine.

2-Amino[1,2,4]triazolo[1,5-c]quinazolines and Derived Novel Heterocycles: Syntheses and Structure-Activity Relationships of Potent Adenosine Receptor Antagonists.

2-Amino[1,2,4]triazolo[1,5-c]quinazolines were identified as potent adenosine receptor (AR) antagonists. Synthetic strategies were devised to gain access to a broad range of derivatives including novel polyheterocyclic compounds. Potent and selective A3 AR antagonists were discovered, including 3,5-diphenyl[1,2,4]triazolo[4,3-c]quinazoline (17, Ki human A3 AR 1.16 nm) and 5'-phenyl-1,2-dihydro-3'H-spiro[indole-3,2'-[1,2,4]triazolo[1,5-c]quinazolin]-2-one (20, Ki human A3 AR 6.94 nm). In addition, multitarget antagonists were obtained, such as the dual A1 /A3 antagonist 2,5-diphenyl[1,2,4]triazolo[1,5-c]quinazoline (13 b, Ki human A1 AR 51.6 nm, human A3 AR 11.1 nm), and the balanced pan-AR antagonists 5-(2-thienyl)[1,2,4]triazolo[1,5-c]quinazolin-2-amine (11 c, Ki human A1 AR 131 nm, A2A AR 32.7 nm, A2B AR 150 nm, A3 AR 47.5 nm) and 9-bromo-5-phenyl[1,2,4]triazolo[1,5-c]quinazolin-2-amine (11 q, Ki human A1 AR 67.7 nm, A2A AR 13.6 nm, A2B AR 75.0 nm, A3 AR 703 nm). In many cases, significantly different affinities for human and rat receptors were observed, which emphasizes the need for caution in extrapolating conclusions between different species.

Ghrelin gene products rescue cultured adult rat hippocampal neural stem cells from high glucose insult.

Adult hippocampal neurogenesis is decreased in type 2 diabetes, and this impairment appears to be important in cognitive dysfunction. Previous studies suggest that ghrelin gene products (acylated ghrelin (AG), unacylated ghrelin (UAG) and obestatin (OB)) promote neurogenesis. Therefore, we hypothesize that ghrelin gene products may reduce the harmful effects of high glucose (HG) on hippocampal neural stem cells (NSCs). The aim of this study was to investigate the role of these peptides on the survival of cultured hippocampal NSCs exposed to HG insult. Treatment of hippocampal NSCs with AG, UAG or OB inhibited HG-induced cell death and apoptosis. Exposure of cells to the growth hormone secretagogue receptor 1a antagonist abolished the protective effects of AG against HG toxicity, whereas those of UAG or OB were preserved. All three peptides attenuated HG-induced decrease in BrdU-labeled and phosphohistone-H3-labeled cells. We also investigated the effects of ghrelin gene products on the regulation of apoptosis at the mitochondrial level. AG, UAG or OB rescued hippocampal NSCs from HG insult by inhibiting intracellular and mitochondrial reactive oxygen species generation and stabilizing mitochondrial transmembrane potential. In addition, cells treated with ghrelin gene products showed an increased Bcl-2 and decreased Bax levels, thereby increasing the Bcl-2/Bax ratio, inhibiting cytochrome c release and preventing caspase-3 activation. Finally, AG-, UAG- or OB-mediated protection was dependent on the activities of adenosine monophosphate-activated protein kinase/uncoupling protein 2 pathway. Our data indicate that ghrelin gene products may act as survival factors that preserve mitochondrial function and inhibit oxidative stress-induced apoptosis.

Reciprocal Catecholamine Changes during Opiate Exposure and Withdrawal.

Dysregulated catecholamine signaling has long been implicated in drug abuse. Although much is known about adaptations following chronic drug administration, little work has investigated how a single drug exposure paired with withdrawal influences catecholamine signaling in vivo. We used fast-scan cyclic voltammetry in freely moving rats to measure real-time catecholamine overflow during acute morphine exposure and naloxone-precipitated withdrawal in two regions associated with the addiction cycle: the dopamine-dense nucleus accumbens (NAc) and norepinephrine-rich ventral bed nucleus of the stria terminalis (vBNST). We compared dopamine transients in the NAc with norepinephrine concentration changes in the vBNST, and correlated release with specific withdrawal-related behaviors. Morphine increased dopamine transients in the NAc, but did not elicit norepinephrine responses in the vBNST. Conversely, dopamine output was decreased during withdrawal, while norepinephrine was released in the vBNST during specific withdrawal symptoms. Both norepinephrine and withdrawal symptoms could be elicited in the absence of morphine by administering naloxone with an α2 antagonist. The data support reciprocal roles for dopamine and norepinephrine signaling during drug exposure and withdrawal. The data also support the allostasis model and show that negative-reinforcement may begin working after a single exposure/withdrawal episode.

The "Ram Effect": A "Non-Classical" Mechanism for Inducing LH Surges in Sheep.

During spring sheep do not normally ovulate but exposure to a ram can induce ovulation. In some ewes an LH surge is induced immediately after exposure to a ram thus raising questions about the control of this precocious LH surge. Our first aim was to determine the plasma concentrations of oestradiol (E2) E2 in anoestrous ewes before and after the “ram effect” in ewes that had a “precocious” LH surge (starting within 6 hours), a “normal” surge (between 6 and 28h) and “late» surge (not detected by 56h). In another experiment we tested if a small increase in circulating E2 could induce an LH surge in anoestrus ewes. The concentration of E2 significantly was not different at the time of ram introduction among ewes with the three types of LH surge. “Precocious” LH surges were not preceded by a large increase in E2 unlike “normal” surges and small elevations of circulating E2 alone were unable to induce LH surges. These results show that the “precocious” LH surge was not the result of E2 positive feedback. Our second aim was to test if noradrenaline (NA) is involved in the LH response to the “ram effect”. Using double labelling for Fos and tyrosine hydroxylase (TH) we showed that exposure of anoestrous ewes to a ram induced a higher density of cells positive for both in the A1 nucleus and the Locus Coeruleus complex compared to unstimulated controls. Finally, the administration by retrodialysis into the preoptic area, of NA increased the proportion of ewes with an LH response to ram odor whereas treatment with the α1 antagonist Prazosin decreased the LH pulse frequency and amplitude induced by a sexually active ram. Collectively these results suggest that in anoestrous ewes NA is involved in ram-induced LH secretion as observed in other induced ovulators.

Protective effects of MCR-1329, a dual α1 and angII receptor antagonist, in mineralocorticoid-induced hypertension.

With the prototypical structures of losartan and prazosin as the axis of our research, MCR-1329 emerged as a potential designed multiple ligand from a series of compounds designed to possess dual antagonistic activity on the α1 and AT1 receptor. After confirming the activity of MCR-1329 in in vitro and acute in vivo models, the present study was undertaken to determine the efficacy of MCR-1329 in a mammalian test system. A rat model of deoxycorticosterone acetate (DOCA)-salt induced renal hypertension following unilateral nephrectomy was utilized to determine the effect of MCR-1329. For mechanistic evaluations, MCR-1329 was evaluated on rat aortic strips in vitro and on rat aortic smooth muscle cells to determine the role of MCR-1329 on phosphoinositide 3 kinase (PI3K) signaling. Results of the study showed that MCR-1329 prevents development of arterial hypertension. It was also observed that MCR-1329 upheld the intimal structures of major arteries like the thoracic aorta. Acetylcholine (Ach)-mediated relaxation remained intact in arteries from MCR-1329 treated animals. It was observed that MCR-1329 partially prevents Thr-308 phosphorylation of Akt following ligand-mediated receptor stimulation in vascular smooth muscle cells. Addition of LY294002 to the reaction medium caused a near-complete inhibition of Akt-phosphorylation. This suggested that MCR-1329 elicits its antihypertensive role by blocking activation of receptor-mediated PI3K-Akt downstream signaling as well as through preservation of arterial integrity. MCR-1329 has the potential to be evaluated further for clinical development as a potential antihypertensive agent with multiple mechanisms of action.

Effects of A1 receptor agonist/antagonist on spontaneous seizures in pilocarpine-induced epileptic rats.

Adenosine is an endogenous anticonvulsant that activates pre- and postsynaptic adenosine A1 receptors. A1 receptor agonists increase the latency for the development of seizures and status epilepticus following pilocarpine administration. Although hippocampal adenosine is increased in the chronic phase of the pilocarpine model, it is not known whether the modulation of A1 receptors may influence the frequency of spontaneous recurrent seizures (SRS). Here, we tested the hypothesis that the A1 receptor agonist RPia ([R]-N-phenylisopropyladenosine) and the A1 antagonist DPCPX (8-Cyclopentyl-1,3-dipropylxanthine) administered to chronic pilocarpine epileptic rats would respectively decrease and increase the frequency of SRS and hippocampal excitability. Four months after Pilo-induced SE, chronic epileptic rats were video-monitored for the recording of SRS before (basal) and after a 2-week treatment with RPia (25μg/kg) or DPCPX (50μg/kg). Following sacrifice, brain slices were studied with electrophysiology. We found that rats given RPia had a 93% nonsignificant reduction in the frequency of seizures compared with their own pretreatment baseline. In contrast, the administration of DPCPX resulted in an 87% significant increase in seizure rate. Nontreated epileptic rats had a similar frequency of seizures along the study. Corroborating our behavioral data, in vitro recordings showed that slices from animals previously given DPCPX had a shorter latency to develop epileptiform activity, longer and higher DC shifts, and higher spike amplitude compared with slices from nontreated Pilo controls. In contrast, smaller spike amplitude was recorded in slices from animals given RPia. In summary, the administration of A1 agonists reduced hippocampal excitability but not the frequency of spontaneous recurrent seizures in chronic epileptic rats, whereas A1 receptor antagonists increased both.

Caffeine exposure alters adenosine system and neurochemical markers during retinal development.

Evidence points to beneficial properties of caffeine in the adult central nervous system, but teratogenic effects have also been reported. Caffeine exerts most of its effects by antagonizing adenosine receptors, especially A1 and A2A subtypes. In this study, we evaluated the role of caffeine on the expression of components of the adenosinergic system in the developing avian retina and the impact of caffeine exposure upon specific markers for classical neurotransmitter systems. Caffeine exposure (5-30mg/kg by in ovo injection) to 14-day-old chick embryos increased the expression of A1 receptors and concomitantly decreased A2A adenosine receptors expression after 48h. Accordingly, caffeine (30mg/kg) increased [(3) H]-8-cyclopentyl-1,3-dipropylxanthine (A1 antagonist) binding and reduced [(3) H]-ZM241385 (A2A antagonist) binding. The caffeine time-response curve demonstrated a reduction in A1 receptors 6h after injection, but an increase after 18 and 24h. In contrast, caffeine exposure increased the expression of A2A receptors from 18 and 24h. Kinetic assays of [(3) H]-S-(4-nitrobenzyl)-6-thioinosine binding to the equilibrative adenosine transporter ENT1 revealed an increase in Bmax with no changes in Kd , an effect accompanied by an increase in adenosine uptake. Immunohistochemical analysis showed a decrease in retinal content of tyrosine hydroxylase, calbindin and choline acetyltransferase, but not Brn3a, after 48h of caffeine injection. Furthermore, retinas exposed to caffeine had increased levels of phosphorylated extracellular signal-regulated kinase and cAMP-response element binding protein. Overall, we show an invivo regulation of the adenosine system, extracellular signal-regulated kinase and cAMP-response element binding protein function and protein expression of specific neurotransmitter systems by caffeine in the developing retina. The beneficial or maleficent effects of caffeine have been demonstrated by the work of different studies. It is known that during animal development, caffeine can exert harmful effects, impairing the correct formation of CNS structures. In this study, we demonstrated cellular and tissue effects of caffeine's administration on developing chick embryo retinas. Those effects include modulation of adenosine receptors (A1 , A2 ) content, increasing in cAMP response element-binding protein (pCREB) and extracellular signal-regulated kinase phosphorylation (pERK), augment of adenosine equilibrative transporter content/activity, and a reduction of some specific cell subpopulations. ENT1, Equilibrative nucleoside transporter 1.