Cancer Stem Cells are Depolarized Relative to Normal Stem Cells Derived from Human Livers

ABSTRACT Introduction and aim. The inability to distinguish cancer (CSCs) from normal stem cells (NSCs) has hindered attempts to identify safer, more effective therapies for hepatocellular carcinoma (HCC). The aim of this study was to document and compare cell membrane potential differences (PDs) of CSCs and NSCs derived from human HCC and healthy livers respectively and determine whether altered GABAergic innervation could explain the differences. Material and methods. Epithelial cell adhesion molecule (EpCAM) positive stem cells were isolated from human liver tissues by magnetic bead separations. Cellular PDs were recorded by microelectrode impalement of freshly isolated cells. GABAA receptor subunit expression was documented by reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence. Results. CSCs were significantly depolarized (-7.0 ± 1.3 mV) relative to NSCs (-23.0 ± 1.4 mV, p < 0.01). The depolarized state was associated with different GABAA receptor subunit expression profiles wherein phasic transmission, represented by GAGAA α3 subunit expression, was prevalent in CSCs while tonic transmission, represented by GABAA α6 subunit expression, prevailed in NSCs. In addition, GABAA subunits α3, β3, γ3 and δ were strongly expressed in CSCs while GABAA π expression was dominant in NSCs. CSCs and NSCs responded similarly to GABAA receptor agonists (ΔPD: 12.5 ± 1.2 mV and 11.0 ± 3.5 mV respectively). Conclusion. The results of this study indicate that CSCs are significantly depolarized relative to NSCs and these differences are associated with differences in GABAA receptor subunit expression. Together they provide new insights into the pathogenesis and possible treatment of human HCC.

Participation of the GABAergic system in the anesthetic effect of Lippia alba (Mill.) N.E. Brown essential oil

The objective of this study was to identify the possible involvement of the GABAergic system in the anesthetic effect of Lippia alba essential oil (EO). We propose a new animal model using silver catfish (Rhamdia quelen) exposed to an anesthetic bath to study the mechanism of action of EO. To observe the induction and potentiation of the anesthetic effect of EO, juvenile silver catfish (9.30 ± 1.85 g; 10.15 ± 0.95 cm; N = 6) were exposed to various concentrations of L. alba EO in the presence or absence of diazepam [an agonist of high-affinity binding sites for benzodiazepinic (BDZ) sites coupled to the GABA A receptor complex]. In another experiment, fish (N = 6) were initially anesthetized with the EO and then transferred to an anesthetic-free aquarium containing flumazenil (a selective antagonist of binding sites for BDZ coupled to the GABA A receptor complex) or water to assess recovery time from the anesthesia. In this case, flumazenil was used to observe the involvement of the GABA-BDZ receptor in the EO mechanism of action. The results showed that diazepam potentiates the anesthetic effect of EO at all concentrations tested. Fish exposed to diazepam and EO showed faster recovery from anesthesia when flumazenil was added to the recovery bath (12.0 ± 0.3 and 7.2 ± 0.7, respectively) than those exposed to water (9.2 ± 0.2 and 3.5 ± 0.3, respectively). In conclusion, the results demonstrated the involvement of the GABAergic system in the anesthetic effect of L. alba EO on silver catfish.

[Effects of harmane, norharmane and harmine on writhing behavior induced by acetic acid in mice]

The beta-carbolines harmane, harmine and norharmane are the members of Harmala,s alkaloids group [Peganum harmala, Zygophillaceae]. The beta-carboline alkaloids adjoined to benzodiazepine site of the gamma-aminobutyric acid type A [GABA[A]]. These alkaloids also inhibited cyclooxygenase and lipoxygenase activities. These findings showed that the beta-carbolines should be able to reduce writhing nociception induced by acetic acid- in mice. To assess the effects of acute treatment with harmane, norharmane and harmine on the writhing induced by acetic acid in mice. The experiments were carried out on male BALB/C mice [20-25g]. Intraperitoneal [I.p] injection of acetic acid [0.6%] was performed in order to cause writhing behavior. This behavior was recorded by direct observation for a 30-minutes period. Decrease of writhing count is indicative of an anti-nociception. In order to avoid the possibility of a physicochemical interaction between them, Drugs were administered on opposite sides of peritoned. Intraperitoneal [I.p] injection of Harmane [5-20mg/kg] on 6-9 mice, norharmane [5-15mg/kg] on 8-9 mice and harmine [10-15mg/kg] on 8-9 mice in per group decreased the writhing behavior significantly [P<0.0001, P<0.0003 and P<0.0016, respectively]. The inhibitory effects of the mentioned drugs were antagonized by flumazenil [2 mg/kg]. Effects of harmane, norharmane and harmine on writhing response may be mediated through an inverse agonistic mechanism located in the benzodiazepine receptors

Effect of Withania somnifera Dunal root extract against pentylenetetrazol seizure threshold in mice: possible involvement of GABAergic system.

Withania somnifera (ashwagandha) is a widely used herb in the Ayurvedic system of medicine. The objective of the present study was to elucidate the effect of W. somnifera root extract (Ws) alone or in combination with exogenous gamma-amino butyric acid (GABA), a GABA receptor agonist or with diazepam, a GABA receptor modulator against pentylenetetrazol (PTZ, iv) seizure threshold in mice. Minimal dose of PTZ (iv, mg/kg) needed to induce different phases (myoclonic jerks, generalized clonus and tonic extension) of convulsions were recorded as an index of seizure threshold. Ws (100 or 200 mg/kg, po) increased the PTZ seizure threshold for the onset of tonic extension phase whereas a lower dose (50 mg/kg, po) did not show any effect on the seizure threshold. Co-administration of a sub-effective dose of Ws (50 mg/kg, po) with a sub-protective dose of either GABA (25 mg/kg, ip) or diazepam (0.5 mg/kg, ip) increased the seizure threshold. The results suggested that the anticonvulsant effect of W. somnifera against PTZ seizure threshold paradigm involved the GABAAergic modulation.

Protective effect of Withania somnifera dunal root extract against protracted social isolation induced behavior in rats.

This study investigated the effect of Withania somnifera Dunal (WS) root extract and diazepam in social isolation induced behavior such as anxiety and depression in rats. Rats were isolated for 6 weeks and the assessment of changed behavior were done on elevated plus maze (EPM) and forced swim test (FST). Isolation reared rats spent less time into the open arms on EPM and significantly increased immobility time in FST compared to group housed rats. WS (100, 200 or 500 mg/kg, oral) and diazepam (1 or 2 mg/kg, ip) dose dependently increased the time spent and entries into the open arms on EPM test and showed the anxiolytic activity. Subeffective dose of WS (50 mg/kg, oral) potentiated the anxiolytic action of diazepam (0.5, 1 or 2 mg/kg, ip). WS (100, 200 or 500 mg/kg, oral) also reduced the immobility time in FST, thus showed antidepressant effect in both group housed and social isolates. The investigations support the use of WS as a mood stabilizer in socially isolation behavior in Ayurveda.

Effect of isopregnanolone on rapid tolerance to the anxiolytic effect of ethanol / Influência da isopregnenolona na tolerância rápida ao efeito ansiolítico do etanol

OBJETIVE: It has been shown that neurosteroids can either block or stimulate the development of chronic and rapid tolerance to the incoordination and hypothermia caused by ethanol consumption. The aim of the present study was to investigate the influence of isopregnanolone on the development of rapid tolerance to the anxiolytic effect of ethanol in mice. METHOD: Male Swiss mice were pretreated with isopregnanolone (0.05, 0.10 or 0.20 mg/kg) 30 min before administration of ethanol (1.5 g/kg). Twenty-four hours later, all animals we tested using the plus-maze apparatus. The first experiment defined the doses of ethanol that did or did not induce rapid tolerance to the anxiolytic effect of ethanol. In the second, the influence of pretreatment of mice with isopregnanolone (0.05, 0.10 or 0.20 mg/kg) on rapid tolerance to ethanol (1.5 g/kg) was studied. CONCLUSIONS: The results show that pretreatment with isopregnanolone interfered with the development of rapid tolerance to the anxiolytic effect of ethanol.

OBJETIVO: Estudos prévios têm mostrado que os neuroesteróides podem bloquear ou estimular o desenvolvimento da tolerância rápida e crônica aos efeitos de incoordenação e hipotermia produzidos pelo etanol. O objetivo do presente estudo foi investigar a influência da isopregnenolona sobre o desenvolvimento da tolerância rápida ao efeito ansiolítico do etanol em camundongos. MÉTODO: Camundongos suíços, machos, foram pré-tratados com isopregnenolona (0,05, 0,10 ou 0,20 mg/kg) 30 minutos antes da administração de etanol (1,5 g/kg). Após 24 horas, todos os animais foram testados no labirinto em cruz elevado. O primeiro experimento foi realizado com o intuito de selecionar uma dose de etanol que produzisse tolerância rápida ao efeito ansiolítico do etanol. No segundo experimento, o objetivo foi investigar o efeito da isopregnenolona (ISO; 0,05, 0,10 ou 0,20 mg/kg) sobre a tolerância rápida ao etanol (1,5 g/kg). CONCLUSÕES: Os resultados mostram que o tratamento prévio com isopregnenolona interferiu no desenvolvimento da tolerância rápida ao efeito ansiolítico etanol.

The bradycardic and hypotensive responses to serotonin are reduced by activation of GABA A receptors in the nucleus tractus solitarius of awake rats

We investigated the effects of bilateral injections of the GABA receptor agonists muscimol (GABA A) and baclofen (GABA B) into the nucleus tractus solitarius (NTS) on the bradycardia and hypotension induced by iv serotonin injections (5-HT, 2 æg/rat) in awake male Holtzman rats. 5-HT was injected in rats with stainless steel cannulas implanted bilaterally in the NTS, before and 5, 15, and 60 min after bilateral injections of muscimol or baclofen into the NTS. The responses to 5-HT were tested before and after the injection of atropine methyl bromide. Muscimol (50 pmol/50 nl, N = 8) into the NTS increased basal mean arterial pressure (MAP) from 115 ± 4 to 144 ± 6 mmHg, did not change basal heart rate (HR) and reduced the bradycardia (-40 ± 14 and -73 ± 26 bpm at 5 and 15 min, respectively, vs -180 ± 20 bpm for the control) and hypotension (-11 ± 4 and -14 ± 4 mmHg, vs -40 ± 9 mmHg for the control) elicited by 5-HT. Baclofen (12.5 pmol/50 nl, N = 7) into the NTS also increased basal MAP, but did not change basal HR, bradycardia or hypotension in response to 5-HT injections. Atropine methyl bromide (1 mg/kg body weight) injected iv reduced the bradycardic and hypotensive responses to 5-HT injections. The stimulation of GABA A receptors in the NTS of awake rats elicits a significant increase in basal MAP and decreases the cardiac Bezold-Jarisch reflex responses to iv 5-HT injections.

Acute phenobarbital administration induces hyperalgesia: pharmacological evidence for the involvement of supraspinal GABA-A receptors

The aim of the present study was to determine if phenobarbital affects the nociception threshold. Systemic (1-20 mg/kg) phenobarbital administration dose dependently induced hyperalgesia in the tail-flick, hot-plate and formalin tests in rats and in the abdominal constriction test in mice. Formalin and abdominal constriction tests were the most sensitive procedures for the detection of hyperalgesia in response to phenobarbital compared with the tail-flick and hot-plate tests. The hyperalgesia induced by systemic phenobarbital was blocked by previous administration of 1 mg/kg ip picrotoxin or either 1-2 mg/kg sc or 10 ng icv bicuculline. Intracerebroventricular phenobarbital administration (5 æg) induced hyperalgesia in the tail-flick test. In contrast, intrathecal phenobarbital administration (5 æg) induced antinociception and blocked systemic-induced hyperalgesia in this test. We suggest that phenobarbital may mediate hyperalgesia through GABA-A receptors at supraspinal levels and antinociception through the same kind of receptors at spinal levels

Ethanol, GABA end epilepsy

Ethanol exerts its behavioral effects largely by interacting with receptors to brain neurotransmitters. The molecular mechanisms involving these interactions are still not well known since an ideal model for their study is currently unavailable. In addition, responses to alcohol may vary due to factors such as genetic predisposition, ethanol concentration consumed, and stimuli such as stress, socialization, etc. The chronc consumption of alcohol, similar to that of other drugs such as benzodiazepines and barbiturates, is linked to GABAerigc neurotransmission. GABA is the predominant inhibitory neurotransmitter in the brain. In context of substance abuse, these three drugs first cause a gratifying effect, later tolerance and finally, physical and psychological dependence. If cosumption is interrupted abruptly, a withdrawal syndrome occurs. The Alcohol Withdrawal Syndrome (AWS) is state of hyperexcitability characterized by anxiety, fear, muscular rigidity and tonic-clonic seizures with epileptiform-type charactermental epilepsy models such as "Kindling" or GABA Withdrawal Syndrome (GWS) models. A possible correlation between these models and AWS will allow for a better understanding of the cellular and molecular effects that alcohol exerts on the brain

Hyperalgesic effect induced by barbiturates, midazolam and ethanol: pharmacological evidence for GABA-A receptor involvement

The involvement of GABA-A receptors in the control of nociception was studied using the tail-flick test in rats. Non-hypnotic doses of the barbiturates phenobarbital (5-50 mg/kg), pentobarbital (17-33 mg/kg), and thiopental (7.5-30 mg/kg), of the benzodiazepine midazolam (10 mg/kg) or of ethanol (0.4-1.6 g/kg) administered by the systemic route reduced the latency for the tail-flick response, thus inducing a 'hyperalgesic' state in the animals. In contrast, non-convulsant doses of the GABA-A antagonist picrotoxin (0.12- 1.0 mg/kg) administered systemically induced an increase in the latency for the tail-flick response, therefore characterizing an 'antinociceptive' state. Previous picrotoxin (0.12 mg/kg) treatment abolished the hyperalgesic state induced by effective doses of the barbiturates, midazolam or ethanol. Since phenobarbital, midazolam and ethanol reproduced the described hyperalgesic effect of GABA-A-specific agonists (muscimol, THIP), which is specifically antagonized by the GABA-A antagonist picrotoxin, our results suggest that GABA-A receptors are tonically involved in the modulation of nociception in the rat central nervous system.

Benzodiazepinas, reguladores alostéricos del receptor GABA-A: alcances fisiológicos, farmacológicos y terapéuticos / Benzodiazepines, allosteric modulators of GABA-A receptor: physiologic, pharmacologic and therapeutic features

The molecular basis underlying the anxiolytic, sedative hypnotic properties of the benzodiazepine compounds are examined at the ligh of recent progress in the field of psychopharmacology and molecular biology. The benzodiazepine receptors, originally characterized as the building site of radiolabelled benzodiazepines, are now identified as allosteric domains of a small population of the plethora of putative brain GABA-A receptor complexes. Emphasis is given to the understanding of the molecular biology of the GABA-A receptor complex and the hypothesis of the multiplicity of benzodiazepine receptors. Experiments describing the need of the gamma subunit of the GABA-A receptor for full benzodiazepine action as positive or negative allosteric modulators of the GABA-A receptor complez are analyzed. Likewise, experiments differentiating the barbiturate receptor from that of the benzodiazepines, alcohol and anesthetic agents are discussed ath the light of recent progress in the molecular biology of the GABA-A receptor. It is concluded that the GABA-A receptor complex is regulated at multiple sites, by a variety of drugs. Regarding putative endogenous ligands for the benzodiazepine receptor, evidences are discussed in support of the endozepines and their implications in health and diseases

Effects of high doses of diazepam on carrageenin-induced paw edema in rats

Benzodiazepine (BDZ) receptor sites play a relevant role in immune/ inflammatory reactions. Acute BDZ treatments were shown not only to suppress cell proliferation in rat thymus but also to decrease TNF-alpha, IL-1 and IL-6 release from adult mouse macrophages. In the present investigation the effects of acute (l0.0 and 20.0 mg/kg) and long-term (10.0 mg kg(-1) day(-l), for 21 days) diazepam treatment on carrageenin-induced paw edema were studied in rats. The results showed that acute treatment with high doses of diazepam decreased paw edema volume in a dose-dependent manner, and this effect was observed as early as 1 h after the administration of the 20.0 mg/kg dose and continued until the last measurement was performed (8 h). In contrast, long-term diazepam administration did not modify the phlogistic-induced edema. Taken together, these data show that 1) acute diazepam treatment with high doses decreases the volume of the acute inflammatory paw edema developed by the organism as a response to carrageenininduced injury, and 2) long-term diazepam treatment induces tolerance to this effect. These results are discussed in the light of a possible effect of diazepam on the components of the rat cellular and humoral immune/inflammatory reaction such as T lymphocytes and/or interleukins.

Benzodiazepinas y fármacos afines / Benzodiazepines and benzodiazepine-like compounds

Se revisan las propiedades farmacológicas, indicaciones clínicas, efectos adversos y potencial de abuso y dependencia de los fármacos benzodiazepínicos y se comparan con dos agentes de efecto predominantemente hipnótico -zolpidem y zopiclona-, que si bien difieren en su estructura química, comparten con éstas la acción a nivel del complejo receptor GABA. Se comenta a la luz de reportes recientes en la literatura el potencial de dependencia de estos dos compuestos, como también, la normativa chilena de prescripción y dispensación de benzodiazepinas. La no inclusión bajo el mismo régimen de zolpidem y zopiclona ha producido un marcado aumento en la dispensación sin prescripción médica de estos últimos, y limita los alcances positivos sobre uso racional de fármacos que se perseguía con las nuevas normas

Alcoolismo e benzodiazepínicos / Alcoholism and benzodiazepine

Com o intuito de estudar a açao e os efeitos dos benzodiazepínicos no tratamento do alcoolismo, foi realizado um levantamento bibliográfico sobre o assunto, com destaque para a açao do álcool e dos benzodiazepínicos no SNC, mais especificamente no sistema gabaérgico. Fazendo-se uma correlaçao farmaco-clínica, constatou-se que as síndromes de abstinência, que variam de leves a severas, como delirium tremens, sao tratadas com diversas dosagens de benzodiazepínicos obtendo-se bons resultados. Nao foi necessária, na maioria dos pacientes, a associaçao com outras drogas depressoras e sedativas.

Evidence for benzodiazepine receptor interaction with MK 801 in anxiety related behaviour in rats.

Antianxiety effect of noncompetitive NMDA receptor antagonist MK 801 was investigated on elevated plus-maze paradigm in rats. Interactional effects of MK 801 with the specific anxiolytic agent, diazepam; the anxiogenic beta-carboline agent, FG-7142 and the central BZ receptor antagonist Ro 15-1788 were explored. MK 801 (0.025-0.1 mg/kg) produced anxiolytic effect at all the doses in rats. Effect was potentiated by diazepam in rats. Both, FG-7142 and Ro 15-1788 reversed the effects of MK 801 and diazepam, when these agents were concomitantly administered. The study revealed anxiolytic profile of MK 801 and indicates that NMDA receptors can modulate the benzodiazepine receptor activity in rodents in elevated plus-maze apparatus.

Pharmacological effects of Withania somnifera root extract on GABAA receptor complex.

A methanolic extract of W. somnifera root inhibited the specific binding of [3H]GABA and [35S]TBPS, and enhanced the binding of [3H]flunitrazepam to their putative receptor sites. The extract (5 micrograms) inhibited [3H]GABA binding by 20 +/- 6 per cent whereas a concentration of 1 mg of the extract produced 100 per cent inhibition. The extract (5-100 micrograms) produced 20 +/- 4 to 91 +/- 16 per cent enhancement of [3H]flunitrazepam binding. In functional studies using 36Cl-influx assay in mammalian spinal cord neurons, W. somnifera root extract increased 36Cl-influx in the absence of GABA. This effect on 36Cl-influx was blocked by bicuculline and picrotoxin; and enhanced by diazepam. These results suggest that the W. somnifera extract contains an ingredient which has a GABA-mimetic activity.

GABA/BZ-and NMDA-receptor interaction in digoxin-induced convulsions in rats.

Digoxin (7.5 micrograms icv) induced 'pop-corn' type of convulsions and 100% mortality. The GABA-ergic agents produced varying degree of protection against digoxin-induced neurotoxicity. Diazepam (4 mg/kg) offered significant protection whereas pentobarbital (5 mg/kg) and baclofen (5 mg/kg) markedly reduced per cent mortality, but ethanol (2 g/kg), progabide (50 mg/kg) and muscimol (0.5 mg/kg) as well as GABA (50 mg/kg) could not offer significant protection in doses used. GABA-ergic agonists; GABA, baclofen, diazepam and pentobarbital when administered along with MK-801 (0.5 mg/kg) a non-competitive NMDA antagonist, a potentiation of anticonvulsant action of MK-801 was observed. MK-801 showed potent anticonvulsant profile in dose range (0.25-1 mg/kg) studied. A synergistic influence of Mg2+ and K+ ions on NMDA receptor antagonism was also observed. A role of GABA-ergic facilitation and NMDA antagonism as a potential anticonvulsant approach in digoxin-induced convulsions in rats has been suggested.

Altered response to GABAergic agents following electro and chemo convulsions in mice.

Effects of GABAergic agents and that of electroconvulsive shock (ECS) treatment were studied on bicuculline and picrotoxin (PTX)-induced convulsions in mice. Neither acute nor chronic ECS had any significant effect on bicuculline-induced convulsions, whereas the latency for PTX-induced convulsions was delayed by both acute and chronic ECS. Baclofen treatment delayed significantly the latency for PTX-induced convulsions in animals which were subjected to both acute and chronic ECS, whereas in bicuculline-induced convulsions, it shortened the latency of convulsions 24 hr after acute ECS. Progabide delayed the bicuculline-induced convulsions except in the case of 24 hr after acute ECS and PTX-induced convulsions except in the case of animals treated chronically with ECS. Fengabine showed no significant effect on bicuculline-induced convulsions. However, on PTX-induced convulsions, the latency was delayed in animals not subjected to ECS and in those subjected to chronic ECS. The possible explanations for the alterations in the effect of GABAergic agents following electro and chemo convulsions are (i) differences in the nature of antagonism by bicuculline and PTX, (ii) alterations in receptor sensitivity or number, and (iii) alterations in the levels of endogenous neurotransmitters, the latter two resulting as a result of acute or chronic ECS.