Prazosin blocks the glutamatergic effects of N-methyl-D-aspartic acid on lordosis behavior and luteinizing hormone secretion in the estrogen-primed female rat

We have observed that intracerebroventricular (icv) injection of selective N-methyl-D-aspartic acid (NMDA)-type glutamatergic receptor antagonists inhibits lordosis in ovariectomized (OVX), estrogen-primed rats receiving progesterone or luteinizing hormone-releasing hormone (LHRH). When NMDA was injected into OVX estrogen-primed rats, it induced a significant increase in lordosis. The interaction between LHRH and glutamate was previously explored by us and another groups. The noradrenergic systems have a functional role in the regulation of LHRH release. The purpose of the present study was to explore the interaction between glutamatergic and noradrenergic transmission. The action of prazosin, an alpha1- and alpha2b-noradrenergic antagonist, was studied here by injecting it icv (1.75 and 3.5 µg/6 µL) prior to NMDA administration (1 µg/2 µL) in OVX estrogen-primed Sprague-Dawley rats (240-270 g). Rats manually restrained were injected over a period of 2 min, and tested 1.5 h later. The enhancing effect induced by NMDA on the lordosis/mount ratio at high doses (67.06 ± 3.28, N = 28) when compared to saline controls (6 and 2 µL, 16.59 ± 3.20, N = 27) was abolished by prazosin administration (17.04 ± 5.52, N = 17, and 9.33 ± 3.21, N = 20, P < 0.001 for both doses). Plasma LH levels decreased significantly only with the higher dose of prazosin (1.99 ± 0.24 ng/mL, N = 18, compared to saline-NMDA effect, 5.96 ± 2.01 ng/mL, N = 13, P < 0.05). Behavioral effects seem to be more sensitive to the alpha-blockade than hormonal effects. These findings strongly suggest that the facilitatory effects of NMDA on both lordosis and LH secretion in this model are mediated by alpha-noradrenergic transmission.

The inhibitory role of sympathetic nervous system in the Ca2+-dependent proteolysis of skeletal muscle

Mammalian cells contain several proteolytic systems to carry out the degradative processes and complex regulatory mechanisms to prevent excessive protein breakdown. Among these systems, the Ca2+-activated proteolytic system involves the cysteine proteases denoted calpains, and their inhibitor, calpastatin. Despite the rapid progress in molecular research on calpains and calpastatin, the physiological role and regulatory mechanisms of these proteins remain obscure. Interest in the adrenergic effect on Ca2+-dependent proteolysis has been stimulated by the finding that the administration of β2-agonists induces muscle hypertrophy and prevents the loss of muscle mass in a variety of pathologic conditions in which calpains are activated. This review summarizes evidence indicating that the sympathetic nervous system produces anabolic, protein-sparing effects on skeletal muscle protein metabolism. Studies are reviewed, which indicate that epinephrine secreted by the adrenal medulla and norepinephrine released from adrenergic terminals have inhibitory effects on Ca2+-dependent protein degradation, mainly in oxidative muscles, by increasing calpastatin levels. Evidence is also presented that this antiproteolytic effect, which occurs under both basal conditions and in stress situations, seems to be mediated by β2- and β3-adrenoceptors and cAMP-dependent pathways. The understanding of the precise mechanisms by which catecholamines promote muscle anabolic effects may have therapeutic value for the treatment of muscle-wasting conditions and may enhance muscle growth in farm species for economic and nutritional purposes.

Glucocorticoids, master modulators of the thymic catecholaminergic system?

There is evidence that the major mediators of stress, i.e., catecholamines and glucocorticoids, play an important role in modulating thymopoiesis and consequently immune responses. Furthermore, there are data suggesting that glucocorticoids influence catecholamine action. Therefore, to assess the putative relevance of glucocorticoid-catecholamine interplay in the modulation of thymopoiesis we analyzed thymocyte differentiation/maturation in non-adrenalectomized and andrenalectomized rats subjected to treatment with propranolol (0.4 mg·100 g body weight-1·day-1) for 4 days. The effects of β-adrenoceptor blockade on thymopoiesis in non-adrenalectomized rats differed not only quantitatively but also qualitatively from those in adrenalectomized rats. In adrenalectomized rats, besides a more efficient thymopoiesis [judged by a more pronounced increase in the relative proportion of the most mature single-positive TCRαβhigh thymocytes as revealed by two-way ANOVA; for CD4+CD8- F (1,20) = 10.92, P < 0.01; for CD4-CD8+ F (1,20) = 7.47, P < 0.05], a skewed thymocyte maturation towards the CD4-CD8+ phenotype, and consequently a diminished CD4+CD8-/CD4-CD8+ mature TCRαβhigh thymocyte ratio (3.41 ± 0.21 in non-adrenalectomized rats vs 2.90 ± 0.31 in adrenalectomized rats, P < 0.05) were found. Therefore, we assumed that catecholaminergic modulation of thymopoiesis exhibits a substantial degree of glucocorticoid-dependent plasticity. Given that glucocorticoids, apart from catecholamine synthesis, influence adrenoceptor expression, we also hypothesized that the lack of adrenal glucocorticoids affected not only β-adrenoceptor- but also α-adrenoceptor-mediated modulation of thymopoiesis.

Effect of selective β-adrenoceptor blockade and surgical resection of the celiac-superior mesenteric ganglion complex on delayed liquid gastric emptying induced by dipyrone, 4-aminoantipyrine, and antipyrine in rats

There is evidence for participation of peripheral β-adrenoceptors in delayed liquid gastric emptying (GE) induced in rats by dipyrone (Dp), 4-aminoantipyrine (AA), and antipyrine (At). The present study aimed to determine whether β-adrenoceptors are involved in delayed GE induced by phenylpyrazole derivatives and the role of the prevertebral sympathetic nervous system in this condition. Male Wistar rats weighing 220-280 g were used in the study. In the first experiment rats were intravenously pretreated with vehicle (V), atenolol 30 mg/kg (ATE, β1-adrenergic antagonist), or butoxamine 25 mg/kg (BUT, β2-adrenergic antagonist). In the second experiment, rats were pretreated with V or SR59230A 2 mg/kg (SRA, β3-adrenergic antagonist). In the third experiment, rats were subjected to surgical resection of the celiac-superior mesenteric ganglion complex or to sham surgery. The groups were intravenously treated with saline (S), 240 µmol/kg Dp, AA, or At, 15 min after pretreatment with the antagonists or V and nine days after surgery. GE was determined 10 min later by measuring the percentage of gastric retention (%GR) of saline labeled with phenol red 10 min after gavage. The %GR (means±SE, n=6) values indicated that BUT abolished the effect of Dp (BUT+Dp vs V+Dp: 35.0%±5.1% vs 56.4%±2.7%) and At (BUT+At vs V+At: 33.5%±4.7% vs 52.9%±2.6%) on GE, and significantly reduced (P<0.05) the effect of AA (BUT+AA vs V+AA: 48.0%±5.0% vs 65.2%±3.8%). ATE, SRA, and sympathectomy did not modify the effects of treatments. These results suggest that β2-adrenoceptor activation occurred in delayed liquid gastric emptying induced by the phenylpyrazole derivatives dipyrone, 4-aminoantipyrine, and antipyrine. Additionally, the released neurotransmitter did not originate in the celiac-superior mesenteric ganglion complex.

The alpha2C-adrenoceptor as a neuropsychiatric drug tar-get - PET studies in human subjects

Positron emission tomography imaging has both academic and applied uses in revealing the distribution and density of different molecular targets in the central nervous system. Following the significant progress made with the dopamine D2 receptor, advances have been made in developing PET tracers to allow analysis of receptor occupancy of many other receptor types as well as evaluating changes in endogenous synaptic transmitter concentrations of transmitters e.g. serotonin and noradrenaline. Noradrenergic receptors are divided into α1-, α2- and β-adrenoceptor subfamilies, in humans each of which is composed of three receptor subtypes. The α2-adrenoceptors have an important presynaptic auto-inhibitory function on noradrenaline release but they also have postsynaptic roles in modulating the release of other neurotransmitters, such as serotonin and dopamine. One of the subtypes, the α2C-adrenoceptor, has been detected at distinct locations in the central nervous system, most notably the dorsal striatum. Several serious neurological conditions causing dementia, Alzheimer’s disease and Parkinson’s disease have been linked to disturbed noradrenergic signaling. Furthermore, altered noradrenergic signaling has also been implicated in conditions like ADHD, depression, anxiety and schizophrenia. In order to benefit future research into these central nervous system disorders as well as being useful in the clinical development of drugs affecting brain noradrenergic neurotransmission, validation work of a novel tracer for positron emission tomography studies in humans was performed. Altogether 85 PET imaging experiments were performed during four separate clinical trials. The repeatability of [11C]ORM-13070 binding was tested in healthy individuals, followed by a study to evaluate the dose-dependent displacement of [11C]ORM-13070 from α2C-adrenoceptors by a competing ligand, and the final two studies examined the sensitivity of [11C]ORM-13070 binding to reflect changes in endogenous noradrenaline levels. The repeatability of [11C]ORM-13070 binding was very high. The binding properties of the tracer allowed for a reliable estimation of α2C-AR occupancy by using the reference tissue ratio method with low test-retest variability. [11C]ORM-13070 was dose-dependently displaced from its specific binding sites by the subtype-nonselective α2-adrenoceptor antagonist atipamezole, and thus it proved suitable for use in clinical drug development of novel α2C-adrenoceptor ligands e.g. to determine the best doses and dosing intervals for clinical trials. Convincing experimental evidence was gained to support the suitability of [11C]ORM-13070 for detecting an increase in endogenous synaptic noradrenaline in the human brain. Tracer binding in the thalamus tended to increase in accordance with reduced activity of noradrenergic projections from the locus coeruleus, although statistical significance was not reached. Thus, the investigation was unable to fully validate [11C]ORM-13070 for the detection of pharmacologically evoked reductions in noradrenaline levels.

Pro-fibrotic role of ERK3-MK5 during pressure-overload induced cardiac hypertrophy

Il y a 4 isoforme de p38 : α, β, δ, and γ. MK5, à l'origine identifié comme étant un régulateur de PRAK (Regulated/Activated Protein Kinase), est maintenant connu pour être activée par la protéine kinase p38 (qui est un mitogène activé par la protéine kinase, MAPK). Cette dernière est impliquée dans les mécanismes de fibrose et d'apoptose pendant l'hypertrophie cardiaque. De plus, MK5 est également activée par les MAPKs atypiques; ERK3 et ERK4. Bien qu’elles soient fortement exprimées dans le coeur, le rôle physiologique de MK5 et ERK3 demeure inconnu. Par conséquent, nous avons étudié l'effet de la constriction aortique transversale (TAC) – induisant un surcharge chronique de pression chez les souris hétèrozygotes knockout pour MK5 (MK5+/-) ou ERK3 (ERK3+/-) et pour leurs types sauvages (MK5+/+ et ERK3+/+). Deux sem post-TAC; le ratio de poids du coeur/poids corporel a été augmenté chez les 2 souris MK5+/- et MK5+/+. L'échocardiographie de la trans-thoracique démontre que la surcharge de pression a altéré la fonction diastolique du ventricule gauche chez MK5+/+, mais pas chez la souris MK5+/-. De plus, nous avons observé moins de dépôt de collagène, évalué par une coloration au trichrome de Masson, 2 et 3 sem post-TAC chez les souris MK5+/-. Parallèlement, le niveau de l’ARNm de collagène type1 alpha-1 a été significativement diminué dans les coeurs des souris MK5+/-, 2 et 3 sem post-TAC. De même, ERK3, mais pas ERK5 ni p38α, co-IP avec MK5 dans les 2 modèles des coeurs TAC; aigus ou chroniques. En revanche, l’ajout exogénique de GST-MK5 a abaissé ERK4 et p38α, mais pas ERK3 dans les lysâtes de coeur de souris. Par contre, GST-ERK3 et GST-p38α ne démontrent aucune co-IP avec MK5. Ces données suggèrent que dans le coeur seul ERK3, et non ERK4 ou p38α, est capable d’interagir avec, et réguler MK5. A niveau physiologique MK5 interagit entièrement avec ERK3 et par conséquent MK5 n’est pas disponible pour lier les protéines exogéniques. Les souris hétérozygotes pour ERK3 (ERK3+/-) ont également démontré une réduction ou une absence de collagène et une faible expression d’ARNm du collagène type1 alpha1, 3 sem post-TAC. Ces résultats démontrent un important rôle pro-fibrotique de la signalisation MK5-ERK3 pendant une surcharge chronique de pression.Nous avons également démontré 5 variant d'épissage de (MK5.1-5), y compris la forme originale (MK5.1). MK5.2 et MK5.5 subissent une délétion de 6 paires de base dans l’exon 12 : MK5.3 manque l'exon 12 : MK5.4 et MK5.5 manquent les exons 2-6. L'expression des ARNm des différents variant d'épissage a été vérifiée par PCR en temps réel (qPCR). Bien que l’expression est ubiquitaire, l'abondance relative de chaque variant était tissu-spécifique (coeur, rein, pancréas, muscle squelettique, poumon, foie, et cerveau). En plus, l'abondance relative des variant d’épissage varie pendant la surcharge de pression et le développement postnatal du coeur. En outre, l'immunofluorescence a indiqué que MK5.1-5.3 se localise au noyau alors que MK5.4-5.5 est situé au niveau cytoplasmic dans les cellules HEK 293 non stimulées. Suite à une stimulation avec l'anisomycin, un activateur de p38 MAPK, MK5.1-5.3 se translocalise du noyau au cytoplasme alors qu’une petite fraction de MK5.4-5.5 translocalise vers le noyau. Ces variant d'épissage peuvent diversifier la signalisation de MK5-ERK3 dans coeur, mais leur rôle exact oblige des recherches supplémentaires. Excepté l’isoforme δ, toutes les isoformes de p38 sont exprimées dans le coeur et la forme α est considérée comme étant l'isoforme dominante. L’analyse par qPCR et immunobuvardage de type western ont démontré que p38α et p38γ sont les deux isoformes prédominantes alors que p38β et p38δ sont exprimées aux mêmes niveaux dans le coeur de rat adulte. L'immunofluorescence a démontré que p38α et p38γ se trouvent dans le cytoplasme et le noyau. Cependant, suite à la surcharge par TAC, p38γ s'est accumulé dans noyau tandis que la distribution de p38α est demeurée inchangée. Ainsi, l'abondance de p38γ et sa translocalisation nucléaire suite à la surcharge de pression indique un rôle potentiel dans l'expression génique pendant le remodelage cardiaque. En conclusion, nous avons mis en évidence pour la première fois un rôle pro-fibrotique pour la signalisation MK5-ERK3 pendant une surcharge chronique de pression. D'ailleurs, les niveaux comparables d'expression de p38γ avec p38α, et la localisation différentielle de p38γ pendant la surcharge aiguë ou chronique de pression suggèrent différents rôles possibles pour ces isoformes pendant le remodelage hypertrophique cardiaque.

Effets neurophysiologiques de la stimulation du nerf vague : implication dans le traitement de la dépression résistante et optimisation des paramètres de stimulation

La dépression est une pathologie grave qui, malgré de multiples stratégies thérapeutiques, demeure résistante chez un tiers des patients. Les techniques de stimulation cérébrale sont devenues une alternative intéressante pour les patients résistants à diverses pharmacothérapies. La stimulation du nerf vague (SNV) a ainsi fait preuve de son efficacité en clinique et a récemment été approuvée comme traitement additif pour la dépression résistante. Cependant, les mécanismes d’action de la SNV en rapport avec la dépression n’ont été que peu étudiés. Cette thèse a donc eu comme premier objectif de caractériser l’impact de la SNV sur les différents systèmes monoaminergiques impliqués dans la pathophysiologie de la dépression, à savoir la sérotonine (5-HT), la noradrénaline (NA) et la dopamine (DA), grâce à l’utilisation de techniques électrophysiologiques et de la microdialyse in vivo chez le rat. Des études précliniques avaient déjà révélé qu’une heure de SNV augmente le taux de décharge des neurones NA du locus coeruleus, et que 14 jours de stimulation sont nécessaires pour observer un effet comparable sur les neurones 5-HT. Notre travail a démontré que la SNV modifie aussi le mode de décharge des neurones NA qui présente davantage de bouffées, influençant ainsi la libération terminale de NA, qui est significativement augmentée dans le cortex préfrontal et l’hippocampe après 14 jours. L’augmentation de la neurotransmission NA s’est également manifestée par une élévation de l’activation tonique des récepteurs postsynaptiques α2-adrénergiques de l’hippocampe. Après lésion des neurones NA, nous avons montré que l’effet de la SNV sur les neurones 5-HT était indirect, et médié par le système NA, via l’activation des récepteurs α1-adrénergiques présents sur les neurones du raphé. Aussi, tel que les antidépresseurs classiques, la SNV augmente l’activation tonique des hétérorécepteurs pyramidaux 5-HT1A, dont on connait le rôle clé dans la réponse thérapeutique aux antidépresseurs. Par ailleurs, nous avons constaté que malgré une diminution de l’activité électrique des neurones DA de l’aire tegmentale ventrale, la SNV induit une augmentation de la DA extracellulaire dans le cortex préfrontal et particulièrement dans le noyau accumbens, lequel joue un rôle important dans les comportements de récompense et l’hédonie. Un deuxième objectif a été de caractériser les paramètres optimaux de SNV agissant sur la dépression, en utilisant comme indicateur le taux de décharge des neurones 5-HT. Des modalités de stimulation moins intenses se sont avérées aussi efficaces que les stimulations standards pour augmenter l’activité électrique des neurones 5-HT. Ces nouveaux paramètres de stimulation pourraient s’avérer bénéfiques en clinique, chez des patients ayant déjà répondu à la SNV. Ils pourraient minimiser les effets secondaires reliés aux périodes de stimulation et améliorer ainsi la qualité de vie des patients. Ainsi, ces travaux de thèse ont caractérisé l’influence de la SNV sur les trois systèmes monoaminergiques, laquelle s’avère en partie distincte de celle des antidépresseurs classiques tout en contribuant à son efficacité en clinique. D’autre part, les modalités de stimulation que nous avons définies seraient intéressantes à tester chez des patients recevant la SNV, car elles devraient contribuer à l’amélioration des bénéfices cliniques de cette thérapie.

Clonidina para el Tratamiento del Dolor Oncológico: una Revisión Sistemática de la Literatura

El dolor oncológico representa una de las principales causas de dolor crónico, siendo los opioides la primera línea de manejo, sin embargo 10% de los pacientes requieren estrategias analgésicas multimodales. La eficacia analgésica de la clonidina como coadyuvante ha sido demostrada para diversos modelos de dolor. Sin embargo no hay revisiones sistemáticas que validen su eficacia y seguridad en dolor crónico oncológico. Se realizó una revisión sistemática de la literatura a noviembre 26 de 2012, encontrando 15 trabajos (12 reportes de caso y tres ensayos clínicos controlados), n=138 pacientes. La intervención tuvo una eficacia entre 44,7 y 100%, mostrando mayor beneficio en pacientes con componente de dolor neuropático. La adición de clonidina fue bien tolerada, siendo la sedación y la disminución en tensión arterial y frecuencia cardiaca los efectos secundarios más frecuentes, con relación dosis dependiente, de resolución espontánea y en ninguno de los casos se documentó lesión secundaria en los pacientes. La vía de administración más frecuente fue neuroaxial (intratecal y peridural). La revisión sistemática no fue susceptible de metaanálisis por la heterogeneidad clínica de los estudios. Los resultados obtenidos sugieren que la adición de clonidina puede ser una opción terapeútica eficaz y segura en los pacientes con dolor crónico oncológico severo refractario a opioides a altas dosis asociado o no a infusión neuroaxial de anestésico local, en especial en presencia de componente neuropático. Sin embargo se identificó la necesidad de un mayor número de ensayos clínicos controlados aleatorios que permitan establecer conclusiones definitivas.

Synthesis of FimH receptor-active manno-oligosaccharides by reverse hydrolysis using alpha-mannosidases from Penicillium citrinum, Aspergillus phoenicis and almond

Recombinant Penicillium citrinum alpha-1,2-mannosidase, expressed in Aspergillus oryzae, was employed to carry out regioselective synthesis of alpha-D-mannopyranosyl-(1-->2)-D-mannose. Yields (w/w) of 16.68% disaccharide, 3.07% trisaccharide and 0.48% tetrasaccharide were obtained, with alpha1-->2 linkages present at 98.5% of the total linkages formed. Non-specific alpha-mannosidase from almond was highly efficient in reverse hydrolysis and oligosaccharide yields of 45-50% were achieved. The products of the almond mannosidase were a mixture of disaccharides (30.75%, w/w), trisaccharides (12.26%, w/w) and tetrasaccharides (1.89%, w/w) with 1-->2, 1-->3 and 1-->6 isomers. alpha-1,2-linkage specific mannosidase from P. citrinum and alpha-1,6-linkage-specific mannosidase from Aspergillus phoenicis were used in combination to hydrolyse the respective linkages from the mixture of isomers, resulting in alpha-D-mannopyranosyl-(1-->3)-D-mannose in 86.4% purity. The synthesised oligosaccharides can potentially inhibit the adhesion of pathogens by acting as 'decoys' of receptors of type-1 fimbriae carried by enterobacteria.

G protein ss gamma subunits mediate presynaptic inhibition of transmitter release from rat superior cervical ganglion neurones in culture

The activation of presynaptic G protein-coupled receptors (GPCRs) is widely reported to inhibit transmitter release; however, the lack of accessibility of many presynaptic terminals has limited direct analysis of signalling mediators. We studied GPCR-mediated inhibition of fast cholinergic transmission between superior cervical ganglion neurones (SCGNs) in culture. The adrenoceptor agonist noradrenaline (NA) caused a dose-related reduction in evoked excitatory postsynaptic potentials (EPSPs). NA-induced EPSP decrease was accompanied by effects on the presynaptic action potential (AP), reducing AP duration and amplitude of the after-hyperpolarization (AHP), without affecting the pre- and postsynaptic membrane potential. All effects of NA were blocked by yohimbine and synaptic transmission was reduced by clonidine, consistent with an action at presynaptic alpha 2-adrenoceptors. NA-induced inhibition of transmission was sensitive to pre-incubation of SCGNs with pertussis toxin (PTX), implicating the involvement of G alpha(i)/(o)beta y subunits. Expression of G alpha transducin, an agent which sequesters G protein beta gamma (G beta y) subunits, in the presynaptic neurone caused a time-dependent attenuation of NA-induced inhibition. Injection of purified G beta gamma subunits into the presynaptic neurone inhibited transmission, and also reduced the AHP amplitude. Furthermore, NA-induced inhibition was occluded by pre-injection of G beta gamma subunits. The Ca2+ channel blocker Cd2+ mimicked NA effects on transmitter release. Cd2+, NA and G beta gamma subunits also inhibited somatic Ca2+ current. In contrast to effects on AP-evoked transmitter release, NA had no clear action on AP-independent EPSPs induced by hypertonic solutions. These results demonstrate that G beta gamma subunits functionally mediate inhibition of transmitter release by alpha 2-adrenoceptors and represent important regulators of synaptic transmission at mammalian presynaptic terminals.

Adrenergic receptor stimulation of the mitogen-activated protein kinase cascade and cardiac hypertrophy.

Phenylephrine and noradrenaline (alpha-adrenergic agonism) or isoprenaline (beta-adrenergic agonism) stimulated protein synthesis rates, increased the activity of the atrial natriuretic factor gene promoter and activated mitogen-activated protein kinase (MAPK). The EC50 for MAPK activation by noradrenaline was 2-4 microM and that for isoprenaline was 0.2-0.3 microM. Maximal activation of MAPK by isoprenaline was inhibited by the beta-adrenergic antagonist, propranolol, whereas the activation by noradrenaline was inhibited by the alpha1-adrenergic antagonist, prazosin. FPLC on a Mono-Q column separated two peaks of MAPK (p42MAPK and p44MAPK) and two peaks of MAPK-activating activity (MEK) activated by isoprenaline or noradrenaline. Prolonged phorbol ester exposure partially down-regulated the activation of MAPK by noradrenaline but not by isoprenaline. This implies a role for protein kinase C in MAPK activation by noradrenaline but not isoprenaline. A role for cyclic AMP in activation of the MAPK pathway was eliminated when other agonists that elevate cyclic AMP in the cardiac myocyte did not activate MAPK. In contrast, MAPK was activated by exposure to ionomycin, Bay K8644 or thapsigargin that elevate intracellular Ca2+. Furthermore, depletion of extracellular Ca2+ concentrations with bis-(o-aminophenoxy)ethane-NNN'N'-tetra-acetic acid (BAPTA) or blocking of the L-type Ca2+ channel with nifepidine or verapamil inhibited the response to isoprenaline without inhibiting the responses to noradrenaline. We conclude that alpha- and beta-adrenergic agonists can activate the MEK/MAPK pathway in the heart by different signalling pathways. Elevation of intracellular Ca2+ rather than cyclic AMP appears important in the activation of MAPK by isoprenaline in the cardiac myocyte.

Phenylephrine promotes phosphorylation of Bad in cardiac myocytes through the extracellular signal-regulated kinases 1/2 and protein kinase A.

Studies in non-cardiomyocytic cells have shown that phosphorylation of the Bcl-2 family protein Bad on Ser-112, Ser-136 and Ser-155 decreases its pro-apoptotic activity. Both phenylephrine (100 microM) and the cell membrane-permeating cAMP analog, 8-(4-chlorophenylthio)-cAMP (100 microM), protected against 2-deoxy-D-glucose-induced apoptosis in neonatal rat cardiac myocytes as assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). In cardiac myocytes, phenylephrine primarily stimulates the alpha-adrenoceptor, but, at high concentrations (100 microM), it also increases the activity of the cAMP-dependent protein kinase, protein kinase A (PKA) through the beta-adrenoceptor. Phenylephrine (100 microM) promoted rapid phosphorylation of Bad(Ser-112) and Bad(Ser-155), though we were unable to detect phosphorylation of Bad(Ser-136). Phosphorylation of Bad(Ser-112) was antagonized by either prazosin or propranolol, indicating that this phosphorylation required stimulation of both alpha(1)- and beta-adrenoceptors. Phosphorylation of Bad(Ser-155) was antagonized only by propranolol and was thus mediated through the beta-adrenoceptor. Inhibitor studies and partial purification of candidate kinases by fast protein liquid chromatography showed that the p90 ribosomal S6 kinases, p90RSK2/3 [which are activated by the extracellular signal-regulated kinases 1 and 2 (ERK1/2)] directly phosphorylated Bad(Ser-112), whereas the PKA catalytic subunit directly phosphorylated Bad(Ser-155). However, efficient phosphorylation of Bad(Ser-112) also required PKA activity. These data suggest that, although p90RSK2/3 phosphorylate Bad(Ser-112) directly, phosphorylation of this site is enhanced by phosphorylation of Bad(Ser-155). These phosphorylations potentially diminish the pro-apoptotic activity of Bad and contribute to the cytoprotective effects of phenylephrine and 8-(4-chlorophenylthio)-cAMP.

Adenosine modulates alpha2-adrenergic receptors through a phospholipase C pathway in brainstem cell culture of rats

Adenosine acts in the nucleus tractus solitarii (NTS), one of the main brain sites related to cardiovascular control. In the present study we show that A(1) adenosine receptor (A(1R)) activation promotes an increase on alpha(2)-adrenoceptor (Alpha(2R)) binding in brainstem cell culture from newborn rats. We investigated the intracellular cascade involved in such modulatory process using different intracellular signaling molecule inhibitors as well as calcium chelators. Phospholipase C, protein kinase Ca(2+)-dependent, IP(3) receptor and intracellular calcium were shown to participate in A(1R)/Alpha(2R) interaction. In conclusion, this result might be important to understand the role of adenosine within the NTS regarding autonomic cardiovascular control. (C) 2009 Elsevier B.V. All rights reserved.

Sympathetic outflow activates the venom gland of the snake Bothrops jararaca by regulating the activation of transcription factors and the synthesis of venom gland proteins

The venom gland of viperid snakes has a central lumen where the venom produced by secretory cells is stored. When the venom is lost from the gland, the secretory cells are activated and new venom is produced. The production of new venom is triggered by the action of noradrenaline on both alpha(1)- and beta-adrenoceptors in the venom gland. In this study, we show that venom removal leads to the activation of transcription factors NF kappa B and AP-1 in the venom gland. In dispersed secretory cells, noradrenaline activated both NF kappa B and AP-1. Activation of NF kappa B and AP-1 depended on phospholipase C and protein kinase A. Activation of NF kappa B also depended on protein kinase C. Isoprenaline activated both NF kappa B and AP-1, and phenylephrine activated NF kappa B and later AP-1. We also show that the protein composition of the venom gland changes during the venom production cycle. Striking changes occurred 4 and 7 days after venom removal in female and male snakes, respectively. Reserpine blocks this change, and the administration of alpha(1)- and beta-adrenoceptor agonists to reserpine-treated snakes largely restores the protein composition of the venom gland. However, the protein composition of the venom from reserpinized snakes treated with alpha(1)- or beta-adrenoceptor agonists appears normal, judging from SDS-PAGE electrophoresis. A sexual dimorphism in activating transcription factors and activating venom gland was observed. Our data suggest that the release of noradrenaline after biting is necessary to activate the venom gland by regulating the activation of transcription factors and consequently regulating the synthesis of proteins in the venom gland for venom production.

New Perspectives on beta-Adrenergic Mediation of Innate and Learned Fear Responses to Predator Odor

In the present study, we investigated the role of noradrenergic transmission in unconditioned and conditioned responses to predatory threats. First, we examined the effects of systemically injected beta-blockers on unconditioned and contextual conditioned response to cat odor. The centrally acting beta-blocker (propranolol) was able to impair unconditioned responses, as well as the acquisition of the contextual fear to cat odor; however, the peripherally acting (nadolol) was not effective. Next, we examined the neural substrate underlying the noradrenergic modulation of the defensive response to cat odor and focused on the dorsal premammillary nucleus (PMd), because it represents the hypothalamic site most responsive to predatory threats and, at the same time, presents a dense plexus of noradrenergic fibers. We were able to see that propranolol significantly reduced PMd-Fos expression in response to cat odor and that beta-adrenoceptor blockade in the PMd, before cat odor exposure, reduced defensive responses to the cat odor and to the cat odor-related environment. We have also shown that beta-adrenoceptor blockade in the PMd, before the exposure to cat odor-related context, impaired the contextual conditioned responses. Overall, the present results provide convincing evidence suggesting that central noradrenergic mediation is critical for the expression of unconditioned and contextual conditioned antipredatory responses. We have further shown that the PMd appears to be an important locus to mediate these beta-adrenoceptor effects.