Comparison of the in vitro binding characteristics of the β-carbolines harman and norharman in rat brain and liver and in bovine adrenal medulla

The in vitro binding of the naturally occurring -carbolines harman and norharman in their tritium-labelled forms to cell membranes from the rat brain and liver and from bovine adrenal medulla was investigated. Displacement of the specific [³H]harman binding in bovine adrenal medulla and rat liver by several -carbolines and monoamine oxidase (MAO) inhibitors revealed the pharmacological profile of a single, high-affinity binding site (K _D 4.92±0.43 nmol/l, B_max 8.47±0.17 pmol/mg protein; adrenal medulla) which corresponded to the active site of MAO type A (MAO-A). Similar characteristics have previously been found for brain tissue from rat, marmoset and pig. In order to determine the temperature dependence of the [³H]harman binding, the K _D and B_max values for rat cerebral cortex were calculated from the results of saturation experiments at 5 temperatures (range: 0°C–37°C). Whereas the B_max values under all conditions were – 4 pmol/mg protein, the K _D values, with increasing temperature, ranged from 3 nmol/l to 30 nmol/l. The calculated linear van't Hoff plot (-In K _D against 1/T) suggested an enthalpy-driven binding of [³H]harman to MAO-A.At least three different [³H]norharman-binding sites were detected. In the rat forebrain, 85% of the specific binding (at about 2 nmol/l of [³H]norharman) can be attributed to a MAO binding site of type B: the binding is displaceable, in nmol/l concentrations by the potent and selective MAO-B inhibitors MDL 72,974A, R(–)-deprenyl and pargyline and, in mol/l concentrations, by S(+)-deprenyl and the potent and selective MAO-A inhibitors clorgyline, harmine, harman, harmaline, brofaromine 5-F--methyltryptamine. After suppression of the MAO binding sites with 1 mol/l clorgyline and mol/l R(–)-deprenyl, a second binding site was found. However, the binding at this site was biphasically displaceable by harman and norharman (Hill-slopes about 0.5 and 0.6, curvilinear Rosenthal plots) suggesting the presence of negative co-operativity or of two binding sites (states). A similar clorgyline/R(–)-deprenyl resistent single (Hill-slopes of displacement by norharman, harman and 6-hydroxy--carboline about unity; linear Rosenthal plots) high affinity binding site (K _D 7.5±2 nmol/l, B_max 130±30 fmol/mg protein) was found in bovine adrenal medullary cell membranes. A third quite different clorgyline/R(–)-deprenyl resistent high-affinity (K _D14 nmol/l) and high-density (B_max 10–30 pmol/mg protein) binding site was detected in the liver. The specific binding at this site was not displaceable by harman or most other substituted -carbolines or by tetrahydro--carbolines, but was displaced by norharman and several newly synthesized 6-substituted aromatic -carbolines (e.g. F-, CH₃-, CH₃O-, HO-). The [³H]norharman binding site in the liver is certainly not identical with any of the binding sites for MAO-inhibitors, benzodiazepines or sigma receptor ligands and is slightly enriched in the microsomal (P₃) fraction whereas most of the specific [³H]harman binding was detected in the crude mitochondrial (P₂) fraction. Correspondence to: T. May at the above address     

GABAA ρ receptor mechanisms in the rat amygdala and its role in the modulation of fear and anxiety

Rationale  

Accumulating evidence for the presence of GABA_A ρ receptors within the amygdala which differ from other members of the GABA_A receptor family in both subunit composition and functional properties has been recently obtained.     

O13 Computer Simulations as Alternatives in Education and in Research： the Sim-and cLabs-Programs and the BioSim Project

Teaching programs of the successfully used in practical While these programs originally ＂Virtual Physiology＂ series （ SimNerv, SimHeart, etc. ） are physiology and pharmacology courses all over the world. y might have been seen as only minor substitutes of the real labs it turned out in the course of teaching that they have major didactic advantages especially because the students are much more active in the virtual than in the animal labs. More recently, an advanced series of virtual computer labs （e. g. cLABs-Neuron） has been developed which also allows to do experiments that would be to difficult for most students to be physically carried out but can be realized in a virtual lab （http：//www. clabs, de/）.     

β-blockers and the risk of incident depression in the elderly

The hypothesis that β-blockers cause depression has been both confirmed and refuted in previous studies. However, in hardly any of these studies, depression was systematically and adequately assessed. The aim of this cohort study was to examine whether β-blockers, in general, highly lipid-soluble, nonselective, or serotonergic receptor-binding β-blockers, are associated with incident depression. Between 1993 and 2005, 5104 elderly persons were followed for incident depressions. Depressions were identified by regular interview and continuous monitoring of medical records. Cases were categorized as clinically relevant depressive symptoms or as depressive syndromes, the latter including Diagnostic and Statistical Manual of Mental Disorders-IV-defined depressive disorders. Pharmacies provided information on filled β-blockers. We used Cox regression with drug use as a time-dependent variable to analyze the data, adjusted for potential demographic covariates, activity of daily living, and (contra)indications for β-blockers. We found that use of β-blockers in general did not convey an increased risk of depressive symptoms (hazard ratio [HR], 0.76; 95% confidence interval [CI], 0.37-1.59) or depressive syndromes (HR, 0.99; 95% CI, 0.53-1.84). Highly lipid-soluble β-blockers, mostly propranolol in our study, were associated with depressive symptoms during the first 3 months of use (HR, 3.31; 95% CI, 1.03-10.6), but not with depressive syndromes. Nonselective or serotonergic receptor affinity was not associated with an increased risk of depressive symptoms or syndromes independent of high lipid solubility. We conclude that β-blockers in general do not convey an increased risk of depression. Lipophilic β-blockers are associated with an increased risk of depressive symptoms.     

Importance of the Locus coeruleus and involvement of α-adrenergic receptors in the post-decapitation reflex in the rat

The latency, duration, hindlimb kick frequency, and total activity components of the post-decapitation reflex (PDR) were measured in the rat using a movement-sensitive transducer. Reduction of brain and spinal cord norepinephrine (NE) caused by neonatal administration of 6-hydroxydopamine (6-OHDA) or 5,7-dihydroxytryptamine, which also reduced brain serotonin, decreased all components of the PDR. Depletion of serotonin or dopamine alone reduced the vigor of the reflex, suggesting that these pathways can influence the PDR but are not essential for the response. Lesions of neurons in the Locus coeruleus, made electrolytically or with 6-OHDA, decreased the intensity of the PDR, with the 6-OHDA-induced lesion being more effective. Depletion of forebrain NE terminals with 6-OHDA did not alter the PDR, consistent with a critical involvement of spinal noradrenergic fibers. The PDR was also decreased by phentolamine and prazosin, but not by propanolol, suggesting an involvement of -adrenergic receptors in the response. This hypothesis was further supported by the finding that the efficacy of a variety of drugs (such as tricyclic antidepressants, phenothiazines, and antihypertensive compounds) for blocking the reflex was apparently related to their affinity for -adrenergic receptors. Thus, the PDR is dependent on noradrenergic fibers in the spinal cord and may provide a simple screen for drugs with suspected -adrenergic blocking properties or for agents that disrupt the function of central noradrenergic fibers.Bruce A. Pappas was a visiting Professor on sabbatical leave from the Department of Psychology, Carleton University, Ottawa, Canada K1S 5B6     

Role of the MPTP in conditioning the heart – translatability and mechanism

Mitochondria have long been known to be the gatekeepers of cell fate. This is particularly so in the response to acute ischaemia‐reperfusion injury (IRI). Following an acute episode of sustained myocardial ischaemia, the opening of the mitochondrial permeability transition pore (MPTP) in the first few minutes of reperfusion, mediates cell death. Preventing MPTP opening at the onset of reperfusion using either pharmacological inhibitors [such as cyclosporin A (CsA) ] or genetic ablation has been reported to reduce myocardial infarct (MI) size in animal models of acute IRI. Interestingly, the endogenous cardioprotective intervention of ischaemic conditioning, in which the heart is protected against MI by applying cycles of brief ischaemia and reperfusion to either the heart itself or a remote organ or tissue, appears to be mediated through the inhibition of MPTP opening at reperfusion. Small proof‐of‐concept clinical studies have demonstrated the translatability of this therapeutic approach to target MPTP opening using CsA in clinical settings of acute myocardial IRI. However, given that CsA is a not a specific MPTP inhibitor, more novel and specific inhibitors of the MPTP need to be discovered – the molecular identification of the MPTP should facilitate this. In this paper, we review the role of the MPTP as a target for cardioprotection, the potential mechanisms underlying MPTP inhibition in the setting of ischaemic conditioning, and the translatability of MPTP inhibition as a therapeutic approach in the clinical setting.

Linked Articles

This article is part of a themed section on Conditioning the Heart – Pathways to Translation. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue‐8

Abbreviations

ANT

adenine nucleotide translocase

CABG

coronary artery bypass graft

CsA

cyclosporin A

CypD

cyclophilin D

Drp1

dynamin‐related protein 1

GSK

glycogen synthase kinase

IPC

ischaemic preconditioning

IPost

ischaemic postconditioning

IRI

ischaemia‐reperfusion injury

LV

left ventricular

MI

myocardial infarct

MitoK_ATP

mitochondrial ATP‐sensitive potassium channel

MPTP

mitochondrial permeability transition pore

OMM

outer mitochondrial membrane

OPA1

optic atrophy 1

PMI

perioperative myocardial injury

PPCI

primary percutaneous coronary intervention

RIC

remote ischaemic conditioning

RISK

reperfusion injury salvage kinase

ROS

reactive oxygen species

SAFE

survivor activating factor enhancement

STEMI

ST segment elevation myocardial infarction

VDAC

voltage‐dependent anion channel

Tables of Links

Purinergic signalling in the lung: important in asthma and COPD?

During the past decade, evidence has accumulated for the involvement of purines and purine receptors in the pathogenesis and pathophysiology of asthma and chronic obstructive pulmonary diseases. In particular, the indirect bronchospasmic and inflammatory actions of purines via the activation and degranulation of mast cells has been extensively studied and reviewed. At present, interest is focused on the impact of purines on pulmonary innervation and the neuroendocrine system, and the potent interaction of the latter with many of the airway components that are essentially altered in asthma and chronic obstructive pulmonary disease. However, data related to these interactions are diverse and not always easily recognised in the literature.     

MRP4 and CFTR in the regulation of cAMP and β-adrenergic contraction in cardiac myocytes

Spatiotemporal regulation of cAMP in cardiac myocytes is integral to regulating the diverse functions downstream of β-adrenergic stimulation. The activities of cAMP phosphodiesterases modulate critical and well-studied cellular processes. Recently, in epithelial and smooth muscle cells, it was found that the multi-drug resistant protein 4 (MRP4) acts as a cAMP efflux pump to regulate intracellular cAMP levels and alter effector function, including activation of the cAMP-stimulated Cl(-) channel, CFTR (cystic fibrosis transmembrane conductance regulator). In the current study we investigated the potential role of MRP4 in regulating intracellular cAMP and β-adrenergic stimulated contraction rate in cardiac myocytes. Cultured neonatal ventricular myocytes were used for all experiments. In addition to wildtype mice, β(1)-, β(2)-, and β(1)/β(2)-adrenoceptor, and CFTR knockout mice were used. MRP4 expression was probed via Western blot, intracellular cAMP was measured by fluorescence resonance energy transfer, while the functional role of MRP4 was assayed via monitoring of isoproterenol-stimulated contraction rate. We found that MRP4 is expressed in mouse neonatal ventricular myocytes. A pharmacological inhibitor of MRP4, MK571, potentiated submaximal isoproterenol-stimulated cAMP accumulation and cardiomyocyte contraction rate via β(1)-adrenoceptors. CFTR expression was critical for submaximal isoproterenol-stimulated contraction rate. Interestingly, MRP4-dependent changes in contraction rate were CFTR-dependent, however, PDE4-dependent potentiation of contraction rate was CFTR-independent. We have shown, for the first time, a role for MRP4 in the regulation of cAMP in cardiac myocytes and involvement of CFTR in β-adrenergic stimulated contraction. Together with phosphodiesterases, MRP4 must be considered when examining cAMP regulation in cardiac myocytes.     

Comparison of the intestinal absorption and bioavailability of γ-tocotrienol and α-tocopherol: in vitro, in situ and in vivo studies

The aim of this work was to compare the intestinal absorption kinetics and the bioavailability of γ-tocotrienol (γ-T3) and α-tocopherol (α-Tph) administered separately as oil solutions to rats in vivo. Also, to explain the significant difference in the oral bioavailability of the compounds: (1) the release profiles using the dynamic in vitro lipolysis model, (2) the intestinal permeability and (3) carrier-mediated uptake by Niemann-Pick C1-like 1 (NPC1L1) transporter were examined. Absolute bioavailability studies were conducted after oral administration of γ-T3 or α-Tph prepared in corn oil to rats. In situ rat intestinal perfusion with ezetimibe (a NPC1L1 inhibitor) was performed to compare intestinal permeability. The in vitro interaction kinetics with NPC1L1 was examined in NPC1L1 transfected cells. While the in vitro release studies demonstrated a significantly higher release rate of γ-T3 in the aqueous phase, the oral bioavailability of α-Tph (36%) was significantly higher than γ-T3 (9%). Consequent in situ studies revealed significantly higher intestinal permeability for α-Tph compared with γ-T3 in rats. Moreover, the NPC1L1 kinetic studies demonstrated higher Vmax and Km values for α-Tph compared with γ-T3. Collectively, these results indicate that intestinal permeability is the main contributing factor for the higher bioavailability of α-Tph. Also, these results emphasize the potentially important role of intestinal permeability in the bioavailability of γ-T3, suggesting that enhancing its permeability would increase its oral bioavailability.     

Kinetics of butyrate metabolism in the normal colon and in ulcerative colitis: the effects of substrate concentration and carnitine on the β-oxidation pathway

Aliment Pharmacol Ther 2011; 34: 526–532

Summary

Background Butyrate, a colonic metabolite of carbohydrates, is considered as the major energy source for the colonic mucosa. An impaired butyrate metabolism has been reported in ulcerative colitis (UC), however, the cause still remains unknown. Aim In the present study, we investigated whether higher butyrate concentrations could normalise the oxidation rate in UC. Furthermore, it was investigated whether carnitine could enhance the butyrate oxidation. Methods Mucosal biopsies from a total of 26 UC patients and 25 controls were incubated with ¹⁴C‐labelled Na‐butyrate and the produced ¹⁴CO₂ was measured. First, the rate of oxidative metabolism was compared at three different concentrations of Na‐butyrate (0.05 mm , 1 mm and 10 mm ). Then, incubations of biopsies were performed with carnitine alone or combined with ATP. Results Overall, butyrate oxidation in UC was significantly lower than that in controls. The maximum rate of butyrate oxidation was achieved in UC and control subjects from 1 mm onwards. Increasing the butyrate concentration to a level to be present in the colonic lumen, i.e. 10 mm , did not increase the rate of butyrate oxidation in UC to the rate observed in controls. Addition of carnitine alone or combined with ATP caused no effects. Conclusions Saturation of butyrate kinetics was achieved from 1 mm in UC and control subjects. The rate of butyrate metabolism was significantly impaired in active ulcerative colitis. The addition of compounds interfering with the β‐oxidation pathway had no effect on the butyrate metabolism in UC.     

β-Glucuronidase and β-glucosidase activity and human fecal water genotoxicity in the presence of probiotic lactobacilli and the heterocyclic aromatic amine IQ in vitro

The aim of the study was to assess the genotoxicity of fecal water (FW) and the activity of fecal enzymes (β-glucuronidase and β-glucosidase) after incubation with 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ) and probiotic lactobacilli: Lb. casei 0900, Lb. casei 0908, and Lb. paracasei 0919. Our results show that the carcinogen IQ greatly increased FW genotoxicity (up to 16.92 ± 3.03 U/mg) and the activity of fecal enzymes (up to even 1.4 ± 0.16 U/mg) in 15 individuals (children, adults and elderly). After incubation with IQ, the activity of β-glucuronidase was reduced by Lactobacillus bacteria by 76.0% (Lb. paracasei 0908) in the FW of children, and by 82.0% (Lb. paracasei 0919) in the elderly; while that of β-glucosidase was reduced by 55.0% in children (Lb. casei 0908) and 90.0% (Lb. paracasei 0919) in elderly subjects. Lactobacilli decreased the genotoxicity of FW after incubation with IQ to the greatest extent in adults (by 64.5%). Probiotic lactobacilli, in the presence of IQ, efficiently inhibits activity of fecal enzymes to the level of control. Genotoxicity inhibition depends on the person's age, its individual microbiota and diet.     

Epidemiology and prevalence of extended-spectrum β-lactamase- and carbapenemase-producing Enterobacteriaceae in humans,animals and the environment in West and Central Africa

Extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E) and carbapenemase-producing Enterobacteriaceae (CPE) are widespread. Here we used the ‘One Health’ approach to determine knowledge gaps on ESBL-E and CPE in West and Central Africa. We searched all articles on ESBL-E and CPE in these African regions published in PubMed, African Journals Online and Google Scholar from 2000 onwards. Among the 1201 articles retrieved, we selected 165 studies (West Africa, 118; Central Africa, 47) with data from 22 of the 26 West and Central Africa countries. Regarding the settings, 136 articles focused only on humans (carriage and/or infection), 6 articles on humans and animals, 13 on animals, 1 on humans and the environment, 8 on the environment and 1 on humans, animals and environments. ESBL-E prevalence ranged from 11–72% in humans and 7–79% in aquatic environments (wastewater). In animals, ESBL-E prevalence hugely varied: 0% in cattle, 11–36% in chickens, 20% in rats, 21–71% in pigs and 32–75% in dogs. The bla_CTX-M-15 gene was the predominant ESBL-encoding gene and was associated with plasmids of incompatibility groups F, H, K, Y, N, I1 and R. CPE were studied only in humans. Class B metallo-β-lactamases (NDM) and class D oxacillinases (OXA-48 and OXA-181) were the most common carbapenemases. Our results show major knowledge gaps, particularly on ESBL and CPE in animals and the environment, that might limit antimicrobial resistance management in these regions. The results also emphasise the urgent need to improve active surveillance programmes in each country and to support antimicrobial stewardship.     

Evidence that LPS-reactive arthritis in rats depends on the glial activity and the fractalkine-TNF-α signaling in the spinal cord

It is known that primary afferent central terminal sensitization can influence peripheral inflammation, however, it remains to be understood whether spinal cord glia can also contribute to this process. Our aim was to investigate the effect of spinal cord glia inhibition on the pathogenesis of LPS-induced knee-joint monoarthritis in rats and also to investigate the role of fractalkine and TNF-α. LPS was injected into the knee-joint previously primed with carrageenan to cause articular incapacitation, edema, synovial leukocyte infiltration, and GFAP and CD11b/c spinal immunoreactivity (glia-IR) increase. Articular edema was more sensitive to the inhibition by intrathecal fluorocitrate and minocycline than nociception and synovial leukocyte content. The higher doses of both drugs were ineffective when given by intraperitoneal route. Corticosteroid synthesis inhibition by aminoglutethimide did not change the glia inhibitors effect. The inhibitory effect of the dorsal root potential inhibitor, furosemide, was not additive to that caused by fluorocitrate and minocycline. Intrathecal anti-fractalkine and anti-TNF-α inhibited edema, nociception, and synovial leukocytes, while fractalkine caused the opposite effects. The fractalkine effect was inhibited by fluorocitrate and anti-TNF-α. Finally, fluorocitrate, minocycline and anti-fractalkine attenuated, but fractalkine increased, GFAP and CD11b/c IR. The evidence reported herein supports the hypothesis that spinal fractalkine release is involved in glia activation, which via the spinal release of TNF-α, seems to be involved in the development and maintenance of this arthritis model. A possible modulation of the dorsal root reflexes is discussed. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.     

Of mouse and man--what is the value of the mouse in predicting gene expression in humans?

The mouse is the most commonly used mammalian species in biomedical research and is widely regarded as a human surrogate species. In this Editorial, Robert Coleman discusses the validity of this assumption and cautions against the unquestioning acceptance of the mouse an experimental model for drug discovery and development.     

Hypertension and its management in countries in Africa and the Middle East,with special reference to the place of β-blockade

Abstract

Background:

The prevalence and clinical consequences of hypertension in countries in Africa and the Middle East have not been studied as well as in other regions.     

Ciomodulatory activities and gerontological pharmacologyof Chinese herbal drugs in the neuroendocrine-immunesystem in experimental models and aged animals

<正> The aging process in man and experimental animals has been subjected to numerous investigations in recent years in China as well as in many other countries. In the past 10 years, we have been interested in the evaluation of Chinese herbal products in the prevention and restoration of senile changes in the immune and related physiological systems, such as the     

Studies on the antinociception effect of melatonin and its mechanism in the rat

The antinociception effect of melatonin（Mel） and its mechanisms were studied on the trauma- pain model in the rat. A trauma - pain model was established in Wistar rats by combining right - hind limb amputation with 50℃ tail - flick test. The effects of Mel on central sensitization were studied through measuring the changes of the expression of c - fos （ an indicator of nociceptive transmission at the spinal level ） , subtance P（SP） and brain - derived neurotrophic factor （BDNF） in the spinal cord using immunohistochemistry. In addition, the contents of nitric oxide （NO） in brain and spinal tissues were also observed. The results showed that the immunoreactivity of BDNF and c - fos in spinal dorsal horn in injuried rats began to increase at the first day,     

Crosstalk between the proteasome system and autophagy in the clearance of α-synuclein

Aim:

A growing body of evidence suggests that α-synuclein accumulation may play an important role in the pathogenesis of Parkinson''s disease. The aim of this study was to investigate the roles of the proteasome and autophagy pathways in the clearance of wild-type and mutant α-synuclein in PC12 cells.

Methods:

PC12 cells overexpressing either wild-type or A30P mutant α-synuclein were treated with the proteasome inhibitor epoxomicin, the macroautophagy inhibitor 3-MA and the macroautophagy activator rapamycin alone or in combination. The cell viability was assessed using MTT assay. Immunofluorescence and Western blot analysis were used to detect the level of α-synuclein, LAMP-2A, E1 activase, and E2 ligase in the cells. Chymotrypsin-like proteasomal activity was measured using a commercial kit.

Results:

When the proteasome and macroautophagy in the wild-type and mutant cells were inhibited with epoxomicin and 3-MA, respectively, the cell viability was significantly decreased, and the α-synuclein level was increased. Both epoxomicin and 3-MA activated the chaperone-mediated autophagy (CMA) by increasing the level of the CMA-limiting enzyme LAMP-2A. Furthermore, 3-MA or epoxomicin significantly decreased chymotrypsin-like proteasomal activity. 3-MA or epoxomicin did not change E1 activase expression in either mutant or wild-type cells, but increased E2 ligase expression, especially when used together. Macroautophagy inducer rapamycin increased the cell viability and reduced epoxomicin-induced α-synuclein accumulation. Interestingly, CMA was also activated by rapamycin.

Conclusion:

Our results demonstrate the existence of complex crosstalk between different forms of autophagy and between autophagy and the proteasome pathway in the clearance of α-synuclein in PC12 cells.     

Do reductions in the tar and nicotine yields of cigarettes help to explain recent reductions in lung cancer rates in young men and women in the United States?

Between 1985 and 2000, lung cancer rates in U.S. men and women aged 35-54 yr have declined. To investigate whether these declines can adequately be explained by changes in smoking prevalence, consumption, and duration, or if changes in tar and nicotine yields also contributed, two model-fitting approaches were used. Both approaches used individual person National Health Interview Survey data on smoking prevalence, age of starting and time of quitting, and national estimates of consumption per smoker and yields. Both approaches compared observed rates (by sex and age) relative to 1985, with those predicted after successively including various smoking variables into the model, making varying allowance for compensation for reduced yield. Approach A was simpler, based on mean smoking statistics estimated separately for current and former smokers. Approach B used the multistage model and individual smoking histories to estimate risk. Both approaches showed observed declines in risk were (except for men aged 35-39 yr) clearly greater than predicted based only on prevalence, consumption, and duration. Including yield generally improved the fit. At younger ages, models assuming substantial compensation (consistent with evidence from studies relating nicotine yield and intake) fitted well, but at age 50-54 yr in both sexes and age 45-49 yr in women, the decline was better fitted by models assuming little compensation. The conclusions were not sensitive to the precise parameter values assumed in the modeling. Interpretation is not straightforward, but the findings suggest declines in yields have contributed to the recent declines in rates in young U.S. men and women.