High throughput virtual screening and molecular dynamics simulation analysis of phytomolecules against BfmR of Acinetobacter baumannii: anti-virulent drug development campaign.

Acinetobacter baumannii is a notorious multidrug resistant bacterium responsible for several hospital acquired infections assisted by its capacity to develop biofilms. A. baumannii BfmR (RstA), a response regulator from the BfmR/S two-component signal transduction system, is the major controller of A. baumannii biofilm development and formation. As a result, BfmR represents a novel target for anti-biofilm treatment against A. baumannii. The discovery of the high-resolution crystal structure of BfmR provides a good chance for computational screening of its probable inhibitors. Therefore, in this study we aim to search new, less toxic, and natural BfmR inhibitors from 8450 phytomolecules available in the Indian Medicinal Plants, Phytochemistry and Therapeutic (IMPPAT) database by analyzing molecular docking against BfmR (PDB ID: 6BR7). Out of these 8450 phytomolecules 6742 molecules were successfully docked with BfmR with the docking score range -6.305 kcal/mol to +5.120 kcal/mol. Structure based-molecular docking (SB-MD) and ADMET (absorption, distribution, metabolism, excretion, & toxicity) profile examination revealed that Norepinephrine, Australine, Calystegine B3, 7,7 A-Diepialexine, and Alpha-Methylnoradrenaline phytocompounds strongly binds to the active site residues of BfmR. Furthermore, molecular dynamics simulation (MDS) studies for 100 ns and the binding free energy (MM/GBSA) analysis elucidated the binding mechanism of Calystegine B3, 7,7 A-Diepialexine, and Alpha-Methylnoradrenaline to BfmR. In summary, these phytocompounds seems to have the promising molecules against BfmR, and thus necessitates further verification by both in vitro and in vivo experiments. HighlightsBfmR plays a key role in biofilm development and exopolysaccharide (EPS) synthesis in A. baumannii.Computational approach to search for promising BfmR inhibitors from IMPAAT database.The lead phytomolecules such as Calystegine B3, 7,7 A-Diepialexine, and Alpha-Methylnoradrenaline displayed significant binding with BfmR active site.The outcome of BfmR binding phytomolecules has broadened the scope of hit molecules validation.Communicated by Ramaswamy H. Sarma.

Habitat odor can alleviate innate stress responses in mice.

Predatory odors, which can induce innate fear and stress responses in prey species, are frequently used in the development of animal models for several psychiatric diseases including post-traumatic stress disorder (PTSD) following a life-threatening event. We have previously shown that odors can be divided into at least three types; odors that act as (1) innate stressors, (2) as innate relaxants, or (3) have no innate effects on stress responses. Here, we attempted to verify whether an artificial odor, which had no innate effect on predatory odor-induced stress, could alleviate stress if experienced in early life as a habitat odor. In the current study, we demonstrated that the innate responses were changed to counteract stress following a postnatal experience. Moreover, we suggest that inhibitory circuits involved in stress-related neuronal networks and the concentrations of norepinephrine in the hippocampus may be crucial in alleviating stress induced by the predatory odor. Overall, these findings may be important for understanding the mechanisms involved in differential odor responses and also for the development of pharmacotherapeutic interventions that can alleviate stress in illnesses like PTSD.

Absence of nigral degeneration in aged parkin/DJ-1/PINK1 triple knockout mice.

Recessively inherited loss-of-function mutations in the parkin, DJ-1, or PINK1 gene are linked to familial cases of early-onset Parkinson's diseases (PD), and heterozygous mutations are associated with increased incidence of late-onset PD. We previously reported that single knockout mice lacking Parkin, DJ-1, or PINK1 exhibited no nigral degeneration, even though evoked dopamine release from nigrostriatal terminals was reduced and striatal synaptic plasticity was impaired. In this study, we tested whether inactivation of all three recessive PD genes, each of which was required for nigral neuron survival in the aging human brain, resulted in nigral degeneration during the lifespan of mice. Surprisingly, we found that triple knockout mice lacking Parkin, DJ-1, and PINK1 have normal morphology and numbers of dopaminergic and noradrenergic neurons in the substantia nigra and locus coeruleus, respectively, at the ages of 3, 16, and 24 months. Interestingly, levels of striatal dopamine in triple knockout mice were normal at 16 months of age but increased at 24 months. These results demonstrate that inactivation of all three recessive PD genes is insufficient to cause significant nigral degeneration within the lifespan of mice, suggesting that these genes may be protective rather than essential for the survival of dopaminergic neurons during the aging process. These findings also support the notion that mammalian Parkin and PINK1 may function in the same genetic pathway as in Drosophila.

Gestational hypoxia alone or combined with restraint sensitizes the hypothalamic-pituitary-adrenal axis and induces anxiety-like behavior in adult male rat offspring.

We have reported that hypoxia affects the hypothalamic-pituitary-adrenal (HPA) axis and behavior by driving the expression of central corticotropin-releasing hormone (CRH) and its receptors in adult mammals, and this effect is modulated by other factors. Here, we address whether or not intermittent hypoxia (IH) or restraint (R) or a combination of both (IH+R) during gestation would result in differential alteration of the HPA axis and behavior of the adult male offspring. Gravid rats were exposed to IH in a hypobaric chamber (10.8% O(2), altitude of 5 km), R, or both, daily for 4 h for 21 days. Control parameters were set at sea level (20.9% O(2)). All the stressors significantly and differentially increased CRH and corticotropin-releasing factor receptor type 1 (CRHR1) expression but decreased corticotropin-releasing factor receptor type 2 (CRHR2) in the paraventricular nucleus of the hypothalamus (PVN), enhanced CRHR1 mRNA and CRHR2 mRNA expression in the anterior pituitary, and increased plasma adrenocorticotropic hormone (ACTH) and corticosterone (CORT) levels and adrenal weight in adult male offspring aged 120 days. Furthermore, norepinephrine (NE) and dopamine (DA) levels significantly increased in the locus coeruleus (LC), while the percentage of entries into the open arms of the elevated-plus maze test (EPM) markedly declined. In all the above effects, the combination-induced effect was stronger than each stressor alone. Confocal imaging showed a rich colocalization of CRHR1 with CRH or urocortin I (Ucn I), and CRHR2 with CRH or urocortin III (Ucn III) in the PVN, and CRHR1 with CRH in the LC in EPM-tested groups. In conclusion, IH or R alone or both in combination during gestation sensitize the HPA axis and induce anxiety-like behavior of the adult male offspring, and the combined effects are significantly great than IH or R alone. The CRH-NE neural circuit between the PVN and LC through CRH receptor driving might partly be involved in the effects. The differential colocalization of CRH with CRHR1 might be the neural basis of these effects.

Estimates of serotonin and norepinephrine transporter inhibition in depressed patients treated with paroxetine or venlafaxine.

Paroxetine and venlafaxine are potent serotonin transporter (SERT) antagonists and weaker norepinephrine transporter (NET) antagonists. However, the relative magnitude of effect at each of these sites during treatment is unknown. Using a novel blood assay that estimates CNS transporter occupancy we estimated the relative SERT and NET occupancy of paroxetine and venlafaxine in human subjects to assess the relative magnitude of SERT and NET inhibition. Outpatient subjects (N=86) meeting criteria for major depression were enrolled in a multicenter, 8 week, randomized, double-blind, parallel group, antidepressant treatment study. Subjects were treated by forced-titration of paroxetine CR (12.5-75 mg/day) or venlafaxine XR (75-375 mg/day) over 8 weeks. Blood samples were collected weekly to estimate transporter inhibition. Both medications produced dose-dependent inhibition of the SERT and NET. Maximal SERT inhibition at week 8 for paroxetine and venlafaxine was 90% (SD 7) and 85% (SD 10), respectively. Maximal NET inhibition for paroxetine and venlafaxine at week 8 was 36% (SD 19) and 60% (SD 13), respectively. The adjusted mean change from baseline (mean 28.6) at week 8 LOCF in MADRS total score was -16.7 (SE 8.59) and -17.3 (SE 8.99) for the paroxetine and venlafaxine-treated patients, respectively. The magnitudes of the antidepressant effects were not significantly different from each other (95%CI -3.42, 4.54, p=0.784). The results clearly demonstrate that paroxetine and venlafaxine are potent SERT antagonists and less potent NET antagonists in vivo. NET antagonism has been posited to contribute to the antidepressant effects of these compounds. The clinical significance of the magnitude of NET antagonism by both medications remains unclear at present.

Postnatal weight gain inhibition does not account for neurobehavioral consequences of neonatal Borna disease virus infection.

Neonatal Borna disease virus (BDV) infection of the rat's brain produces neurodevelopmental damage similar to some pathological and clinical features of human developmental disorders, e.g., autism and schizophrenia. Since BDV-infected rats exhibited an inhibition of postnatal weight gain, the present study sought to evaluate a contribution of nutritional status to virus-induced neurodevelopmental injury. We compared neuroanatomical, neurochemical, and behavioral alterations following neonatal BDV infection and rearing in the oversized litters in Fischer344 rats on postnatal day (PND) 26. Despite a comparable weight gain inhibition, different patterns of brain pathology, alterations in brain monoamine systems, and behavioral deficits were observed in the BDV-infected rats compared to the malnourished rats. While no appreciable cell injury was noted in the brains of the malnourished rats, a significant loss of Purkinje cells (PC) and early signs of degeneration of the hippocampal dentate gyrus were found in the BDV-infected rats. Both neonatal BDV infection and postnatal malnourishment increased tissue concentrations of serotonin [5-hydroxytryptamine (5-HT)] in the hippocampus. In contrast, increased turnover of 5-HT in the cortex and hippocampus and elevated turnover of dopamine (DA) in the striatum were found in the malnourished rats only, suggesting that different pathogenic mechanisms might underlie monoamine disturbances in virus-infected and malnourished rats. The observed dissimilar neuroanatomical and neurochemical abnormalities might explain the different responses to novelty in the BDV-infected and malnourished rats. Compared to the control rats, the BDV-infected rats exhibited novelty-induced hyperactivity, while no differences in locomotion were noted between the control and malnourished rats. Taken together, the present data indicate that virus-associated inhibition of postnatal weight gain is unlikely to account for the major BDV-associated neurodevelopmental alterations that seem to be due to specific effects of neonatal BDV infection.

Short exposure of maturing, bone marrow-derived dendritic cells to norepinephrine: impact on kinetics of cytokine production and Th development.

The information gathered by dendritic cells (DC) during the innate immune response to a pathogen is determinant for the type of adaptive response. Here we show that short-term (3 h) exposure of bone marrow-derived DC to norepinephrine (NE), at the beginning of lipopolysaccharide (LPS) or keyhole limpet hemocyanin (KLH) stimulation hampers IL-12 production and increases IL-10 release. The NE effect was mediated by both beta- and alpha2-adrenergic receptors. The capacity of NE-exposed DC to produce IL-12 upon CD40 cross-linking as well as to stimulate allogeneic T-helper (Th) lymphocytes was reduced. Adoptive transfer of NE-exposed DC induced a Th2 slanted response in vivo. Thus, a brief NE exposure of antigen-stimulated DC seems to limit their Th1 polarizing properties. Noteworthy, the ganglionic blocker pentolinium administered in mice before skin sensitization with fluoroscein isothiocyanate (FITC) could increase the Th1-type response in the draining lymph nodes. Our results suggest that the extent of Th differentiation in the response to an antigen might be influenced by the local sympathetic nervous activity in the early phase of dendritic cell stimulation.

Age-related changes in adrenergic neuroeffector transmission.

In this study we have looked at the effects of ageing on prejunctional control of noradrenergic neurotransmission in the cardiovascular system, in terms of alpha2-adrenoceptors, beta2-adrenoceptors and the noradrenaline re-uptake process. These studies show diminished prejunctional alpha2- and prejunctional beta-adrenoceptor-mediated responsiveness together with diminished noradrenaline re-uptake in rat tissues. The reduced prejunctional alpha2-inhibitory control and reduced re-uptake found in tissues from aged rats is more than likely to outweigh the effects of reduced beta-adrenoceptor facilitation, at least in normal conditions. Hence, assuming that such changes also occur in man, we might expect to find evidence of increased release of noradrenaline from noradrenergic nerves, and this could be reflected in plasma levels of noradrenaline.

Displacement of norepinephrine by alpha-methylnorepinephrine in the nucleus tractus solitarius of the rat.

Rat brain slices from the dorsomedial medulla containing the nucleus tractus solitarius were loaded with [3H]norepinephrine ([3H]NE) for superfusion. Electrical stimulation (3 Hz, 25 mA, 1 min) resulted in fractional release ratios S2/S1 of 0.97 +/- 0.02 in normal Krebs-Henseleit (KH) and 0.93 +/- 0.06 in the presence of 30 microM cocaine. With cocaine in the KH medium, L-alpha-methylnorepinephrine (alpha-MeNE) significantly reduced the [3H]NE release S2/S1 without affecting the basal release ratios. Without cocaine in the KH medium both 0.1 and 1.0 microM alpha-MeNE increased the basal release B2/B1 that was not affected by yohimbine. Prazosin had no effect on the S2/S1 ratio but did attenuate the basal release effects of alpha-MeNE. In low Ca2+ studies where the S2 stimulus was abolished, 1.0 microM alpha-MeNE induced a sharply elevated increase in the B2/B1 ratio. It appears that alpha-MeNE in the presence of the uptake inhibitors reduces presynaptic neurotransmitter release through alpha 2-adrenoceptors, whereas when uptake of the monoamines was not blocked alpha-MeNE was considerably more efficacious as a displacing agent of neurotransmitter.

Limitation of the alpha-methylnorepinephrine hypothesis in the hypotensive effect of alpha-methyldopa.

To test the hypothesis that alpha-methylnorepinephrine (MNE) is the principal active metabolite involved in the hypotensive action of alpha-methyldopa (MD), we determined the relationship between MD's depressor response and tissue levels of MD metabolites in critical sites. After administration of 250 mg/kg MD intraperitoneally to Sprague-Dawley rats (300 +/- 50 g), we studied both heart (left ventricle) and brainstem MD, MNE and endogenous NE levels using HPLC with electrochemical detection. We also measured systolic blood pressure before and during MD (25-250 mg/kg i.p.) treatment using the tail-cuff method. Our results indicate that: (1) peak MD hypotensive response was dose-dependent. (2) Central NE concentration was maximally reduced by 2 hours whereas peripheral NE was maximally reduced by 18 hours. The maximal hypotensive effect was closer to the central peak distribution of MNE than MD. (3) The MD concentrations and NE concentrations in brainstem and heart showed counterclockwise hysteresis while MNE showed clockwise hysteresis. Furthermore, the area of MNE hysteresis in brainstem was larger than that of NE. We conclude that MD's depressor effect can not be completely explained by the assumption that MNE is the sole active metabolite; alternate metabolites or mechanisms would appear to be operative.

Depletion of brainstem epinephrine stores by alpha-methyldopa: possible relation to attenuated sympathetic outflow.

The antihypertensive effect of alpha-methyldopa (MD) is believed to be critically dependent on its ability to deplete endogenous catecholamines or cause the synthesis of false neurotransmitters. We used liquid chromatography with electrochemical detection (LCEC) and negative chemical ionization gas chromatography-mass spectrometry (GC-MS) for quantitation of catecholamines and MD metabolites in rat. MD intraperitoneally (100 mg/kg q12 hr X 12 days), significantly increased alpha-methylnorepinephrine (MNE) in brain (1.02 +/- 0.33 micrograms/g), heart (1.67 +/- 0.57 micrograms/g) and adrenal glands (114.93 +/- 50.47 micrograms/g) Endogenous norepinephrine (NE), epinephrine (E) and dopamine (DA) were reduced. ME levels were 2.19 +/- 0.44 micrograms/g (n = 6) in the adrenal gland but only 99 +/- 26 pg/g (n = 3) in the brainstem. MD-induced endogenous brainstem NE depletion was more than compensated by MNE production, but brainstem E depletion was not compensated for by a stoichiometric production of brainstem ME. We conclude (1) although ME is a metabolite of MD, it is present in extremely low concentrations in brainstem and (2) central epinephrine-containing neurons are depleted of neurotransmitter by MD therapy. If this selective epinephrine depletion occurs in the bulbospinal tract neurons responsible for maintaining sympathetic tone, then this effect could contribute to the antihypertensive effect of MD.

Alpha 2 adrenergic receptors in hyperplastic human prostate: identification and characterization using [3H] rauwolscine.

[3H]Rauwolscine ([3H]Ra), a selective ligand for the alpha 2 adrenergic receptor, was used to identify and characterize alpha 2 adrenergic receptors in prostate glands of men with benign prostatic hyperplasia. Specific binding of [3H]Ra to prostatic tissue homogenates was rapid and readily reversible by addition of excess unlabelled phentolamine. Scatchard analysis of saturation experiments demonstrates a single, saturable class of high affinity binding sites (Bmax = 0.31 +/- 0.04 fmol./microgram. DNA, Kd = 0.9 +/- 0.11 nM.). The relative potency of alpha adrenergic drugs (clonidine, alpha-methylnorepinephrine and prazosin) in competing for [3H]Ra binding sites was consistent with the order predicted for an alpha 2 subtype. The role of alpha 2 adrenergic receptors in normal prostatic function and in men with bladder outlet obstruction secondary to BPH requires further investigation.

The in vitro pharmacology of chloroquine and quinacrine.

The effects of guanethidine, chloroquine and quinacrine on noradrenergic nerves have been compared in vitro using the isolated expansor secundariorum muscle of chicks. The effect of chloroquine on alpha-methyl-noradrenaline uptake by noradrenergic nerve terminals in various tissues were studied. The inhibitory action of guanethidine and quinacrine on noradrenergic nerves appeared to be mediated intraneuronally. The inhibitory action of chloroquine was readily reversible and unaffected by dexamphetamine. Chloroquine caused supersensitivity of the expansor muscle to noradrenaline by blocking its neuronal reuptake since the supersensitivity caused by denervation was not further increased by chloroquine. This was confirmed by the finding that chloroquine inhibited alpha-methylnoradrenaline uptake (Uptake1). Quinacrine did not cause supersensitivity to noradrenaline, possibly due to its direct depressant action on the expansor secundariorum muscle.

Metabolism of carbidopa to alpha-methyldopamine and alpha-methylnorepinephrine in rats.

Recent observations on the central and peripheral actions of carbidopa (CD) combined with our own results with the compound led us to examine its metabolism and effects on brain catecholamines in rats. CD was found to undergo a two-stage N-deamination process in vivo giving rise to alpha-methyldopa (AMD) and alpha-methyldopamine respectively. Further, beta-hydroxylation yielded alpha-methylnorepinephrine. These metabolic products were demonstrated in rat brain with reductions in norepinephrine and 3-methoxy-4-hydroxyphenylglycol, and little effect on dopamine. These results are consistent with the alpha-2 agonist effects of alpha-methylnorepinephrine. The relative formation of alpha-methyldopamine from CD was about 26% of an equivalent dose of AMD. It is concluded that some of the central effects of CD may be mediated by its metabolism to AMD, which readily crosses the blood-brain barrier. Possible implications of the findings are discussed.

Drug interactions and vasoconstrictors used in local anesthetic solutions.

This study examined widely advertised interactions between sympathomimetic amine vasoconstrictors currently used in dental local anesthetic solutions and MAO inhibitors (phenelzine, 5 mg/kg), phenothiazines (chlorpromazine, 2 mg/kg), and tricyclic antidepressants (desipramine, 2 mg/kg). Twelve greyhound dogs premedicated with morphine and anesthetized with urethane and alpha-chloralose were prepared for physiologic recordings. During a control period, the dogs received bolus injections of epinephrine, norepinephrine, and levonordefrin sufficient to construct log-linear dose-response curves for each agent. Commercial anesthetic solutions, with and without the vasoconstrictors, were also used. The dose-response curves were then reproduced 1 hour after the administration of a drug interactant. Cardiovascular responses were not influenced by the coadministration of local anesthetics or by the prior administration of phenelzine. Chlorpromazine ameliorated pressor responses to norepinephrine and levonordephrin and reversed the hypertensive effect of high-dose epinephrine. Desipramine significantly increased vasoconstrictor potencies, particularly those of levonordefrin and norepinephrine, which were multiplied more than sixfold.

Amine transport into chromaffin ghosts. Kinetic measurements of net uptake of biologically and pharmacologically relevant amines using an on-line amperometric technique.

The kinetic parameters for net transport of dopamine, epinephrine, norepinephrine, 5-hydroxytryptamine, S alpha-methyldopamine, R alpha-methyldopamine, and 1R,2S alpha-methylnorepinephrine into highly purified bovine chromaffin ghosts were determined using an on-line amperometric technique. Chromaffin ghosts devoid of endogenous amines were formed from lysis of chromaffin granules under hypotonic conditions, extensive washing of the scattered membranes, followed by resuspension in iso-osmotic media and overnight dialysis. When chromaffin ghosts formed so as to generate and maintain a large delta pH were suspended in 185 mM KCl, 10 mM Hepes at pH 7.0, 37 degrees C, the addition of MgATP resulted in rapid acidification of the intravesicular space, which was maintained at pH 6.0 (+/- 0.1) for over 30 min. Kinetic net amine transport was subsequently measured with a glassy carbon electrode. The initial rates of uptake were found to follow Michaelis-Menten kinetics. Computer based statistical analysis of the data using distribution-free procedures yielded Km (and V) values as follows: in microM (nmol X mg protein-1 X min-1) dopamine, 16.2 (14.0); R-norepinephrine, 32.5 (12.9); R-epinephrine, 35.1 (15.2); 5-hydroxytryptamine, 4.7 (5.1); S alpha-methyldopamine, 17.7 (11.2); R alpha-methyldopamine, 44.2 (9.9); 1R,2S alpha-methylnorepinephrine, 76.5 (12.5). The physiologic and pharmacologic implications of these kinetic parameters are discussed.

Postjunctional alpha-1 and alpha-2 adrenoceptors in the coronaries of the perfused guinea-pig heart.

Prazosin and yohimbine were used to differentiate postjunctional alpha adrenoceptors in the coronaries of the perfused guinea-pig heart. Two postjunctional alpha adrenoceptor subtypes were distinguished by the affinities of the receptor for yohimbine and prazosin. The pA2 for yohimbine were 8.74 against alpha-methylnorepinephrine and 8.98 against BHT-920, and the pA2 for prazosin was 9.84 against phenylephrine. Yohimbine was not very active against the alpha-1 selective agonist as was prazosin against the alpha-2 selective agonists. Alpha-1 and alpha-2 postjunctional adrenoceptors mediate vasoconstriction in the whole coronary bed of the perfused guinea-pig heart.

Changes in brain alpha-adrenergic receptors after alpha-methyldopa administration to spontaneously hypertensive rats.

The hypotensive action of methyldopa has been linked to production of the metabolites methyldopamine and methylnorepinephrine in brain. We have studied the effect of long-term (72 hour) intravenous infusions of methyldopa to awake restrained spontaneously hypertensive rats and normotensive Wistar-Kyoto control animals to look for differences in hypotensive effect, differences in concentrations of natural and alpha-methylated catecholamines, and differences in alpha 1 and alpha 2-adrenergic receptor populations. Results described here indicate that hypertensive rats have a greater reduction in blood pressure and a larger increase in hypothalamic and brain stem methylnorepinephrine concentrations than do the normotensive animals. The methylnorepinephrine concentration reached a plateau value in hypothalamus in both strains while pons and medulla showed progressive, dose-related increases in concentration. These regional and strain differences in the metabolism of alpha-methyldopa suggest that the production of methylnorepinephrine in brain stem nuclei is most correlated with the hypotensive action of methyldopa. alpha 2 Agonist binding (p-amino-clonidine) declined in both hypothalamus and brain stem, and the fall was greater in hypertensive than in normotensive rats. alpha 1-Adrenergic receptor binding (prazosin) was increased, again more in hypertensive than in normotensive rats. The down regulation of alpha 2-adrenergic receptors and the up regulation of alpha 1-adrenergic receptors are compatible with increased alpha 2-adrenergic agonist presynaptic inhibition of catecholamine release with resultant postsynaptic alpha 1-adrenergic receptor supersensitivity. Spontaneously hypertensive rats showed greater methylnorepinephrine production, larger up regulation of alpha 1-adrenergic receptors, and greater down regulation of alpha 2-adrenergic receptors than did the normotensive animals; these changes may be physiological markers for the greater antisympathetic action of methyldopa in hypertensive animals.

Antihypertensive metabolites of alpha-methyldopa.

alpha-Methyldopa acts through a metabolite in the central nervous system. Of the three metabolites most prominently considered as potential mediators of alpha-methyldopa hypotension--alpha-methyldopamine, alpha-methylnorepinephrine (MNE), and alpha-methylepinephrine (ME)--ME is the most potent depressor agent following intravenous infusion into rats, following injection into the lateral ventricle, and following injection into the solitary tract nucleus (NTS). The depressor effect of ME in the NTS is attenuated as effectively by timolol as by yohimbine, while the combination of both timolol and yohimbine completely abolishes the pharmacological activity of ME in the NTS. ME is approximately eightfold more potent than MNE in the NTS and also has a greater susceptibility to timolol attenuation.