Kethireddy V.V. Ananthalakshmi

Anticonvulsant enaminones depress excitatory synaptic transmission in the rat brain by enhancing extracellular GABA levels

1 Enaminones are a novel group of compounds that have been shown to possess anticonvulsant activity in in vivo animal models of seizures. The cellular mechanism by which these compounds produce their anticonvulsant effects is not yet known. This study examined the effects of enaminones on excitatory synaptic transmission. 2 We studied the effects of 3‐(4′‐chlorophenyl)aminocyclohex‐2‐enone (E118), methyl 4‐(4′‐bromophenyl)aminocyclohex‐3‐en‐6‐methyl‐2‐oxo‐1‐oate (E139) and ethyl 4‐(4′‐hydroxyphenyl)aminocyclohex‐3‐en‐6‐methyl‐2‐oxo‐1‐oate (E169) on isolated evoked, glutamate‐mediated excitatory synaptic responses by recording whole‐cell currents and potentials in cells of the nucleus accumbens (NAc) contained in forebrain slices. 3 The anticonvulsant enaminones (E118 and E139), but not E169, depressed NMDA and non‐NMDA receptor‐mediated synaptic responses. The inhibition of the non‐NMDA response was concentration‐dependent (1.0–100 μM) with a maximal depression of ∼−30%. E118 and E139 had similar potencies (EC50=3.0 and 3.5 μM, respectively) in depressing this response but E139 was more efficacious (Emax=−31.3±3.8%) than E118 (Emax=−22.6±1.6%). 4 The excitatory postsynaptic current (EPSC) depression caused by 10 μM E139 (−27.7±3.8%) was blocked by 1 μM CGP55845 (6.3±8.1%), a potent GABAB receptor antagonist. 5 Pretreatment of slices with γ‐vinylGABA and 1‐(2‐(((diphenylmethylene)imino)oxy)ethyl)‐1,2,5,6‐tetrahydro‐3‐pyridine‐carboxylic acid (NO‐711), an irreversible GABA transaminase (GABA‐T) inhibitor and a GABA reuptake blocker, respectively, like the anticonvulsant enaminones, also caused a depression of the evoked EPSC (−38.1±14.1 and −24.1±8.9%, respectively). In the presence of these compounds, E139 did not cause a further depression of the EPSC. Our data suggest that anticonvulsant enaminones cause EPSC depression by enhancing extracellular GABA levels possibly through the inhibition of either GABA reuptake or GABA‐T enzyme, or both.

Dopamine and adenosine mediate substance P‐induced depression of evoked IPSCs in the rat nucleus accumbens in vitro

The major projection cells of the nucleus accumbens (NAc) are under a strong inhibitory influence from GABAergic afferents and depend on afferent excitation to produce their output. We have earlier reported that substance P (SP), a peptide which is colocalized with GABA in these neurons, depresses excitatory synaptic transmission in this nucleus (Kombian, S.B., Ananthalakshmi, K.V.V., Parvathy, S.S. & Matowe, W.C. (2003) J. Neurophysiol., 89, 728–738). In order to better understand the role of this peptide in the synaptic physiology of the NAc, it is important to determine its effects on inhibitory synaptic responses. Using whole‐cell recording in rat forebrain slices, we show here that SP also depresses evoked inhibitory postsynaptic currents (IPSCs) in the NAc via intermediate neuromodulators. SP caused a partially reversible, dose‐dependent decrease in evoked IPSC amplitude. This effect was present without measurable changes in the holding current, input resistance of recorded cells or decay rate (τ) of IPSCs. It was mimicked by a neurokinin‐1 (NK1) receptor‐selective agonist, [Sar9, Met (O2)11]‐SP, and blocked by an NK1 receptor‐selective antagonist, L 732 138. The SP‐induced IPSC depression was prevented by SCH23390, a dopamine D1‐like receptor antagonist and by 8‐cyclopentyltheophylline, an adenosine A1 receptor blocker. Furthermore, the SP effect was also markedly attenuated by exogenous adenosine, dipyridamole, rolipram and barium. These data show that SP, acting on NK1 receptors, depresses inhibitory synaptic transmission indirectly by enhancing extracellular dopamine and adenosine levels. SP therefore acts in the NAc to modulate both excitatory and inhibitory afferent inputs using the same mechanism(s).

Cholecystokinin activates CCKB receptors to excite cells and depress EPSCs in the rat rostral nucleus accumbens in vitro

The peptide cholecystokinin (CCK) is abundant in the rat nucleus accumbens (NAc). Although it is colocalized with dopamine (DA) in afferent terminals in this region, neurochemical and behavioural reports are equally divided as to whether CCK enhances or diminishes DAs actions in this nucleus. To better understand the role of this peptide in the physiology of the NAc, we examined the effects of CCK on excitatory synaptic transmission and tested whether these are dependent on DA and/or other neuromodulators. Using whole‐cell recording in rat forebrain slices containing the NAc, we show that sulphated CCK octapeptide (CCK‐8S), the endogenously active neuropeptide, consistently depolarized cells and depressed evoked excitatory postsynaptic currents (EPSCs) in the rostral NAc. It caused a reversible, dose‐dependent decrease in evoked EPSC amplitude that was accompanied by an increase in the decay constant of the EPSC but with no apparent change in paired pulse ratio. It was mimicked by unsulphated CCK‐8 (CCK‐8US), a CCKB receptor‐selective agonist, and blocked by LY225910, a CCKB receptor‐selective antagonist. Both CCK‐8S and CCK‐8US induced an inward current with a reversal potential around −90 mV that was accompanied by an increase in input resistance and action potential firing. The CCK‐8S‐induced EPSC depression was slightly reduced in the presence of SCH23390 but not in the presence of sulpiride or 8‐cyclopentyltheophylline. By contrast, it was completely blocked by CGP55845, a potent GABAB receptor‐selective antagonist. These results indicate that CCK excites NAc cells directly while depressing evoked EPSCs indirectly, mainly through the release of GABA.

Anti-Inflammatory Action of Angiotensin 1-7 in Experimental Colitis.

Background There is evidence to support a role for angiotensin (Ang) 1–7 in reducing the activity of inflammatory signaling molecules such as MAPK, PKC and SRC. Enhanced angiotensin converting enzyme 2 (ACE2) expression has been observed in patients with inflammatory bowel disease (IBD) suggesting a role in its pathogenesis, prompting this study. Methods The colonic expression/activity profile of ACE2, Ang 1–7, MAS1-receptor (MAS1-R), MAPK family and Akt were determined by western blot and immunofluorescence. The effect of either exogenous administration of Ang 1–7 or pharmacological inhibition of its function (by A779 treatment) was determined using the mouse dextran sulfate sodium model. Results Enhanced colonic expression of ACE2, Ang1-7 and MAS1-R was observed post-colitis induction. Daily Ang 1–7 treatment (0.01–0.06 mg/kg) resulted in significant amelioration of DSS-induced colitis. In contrast, daily administration of A779 significantly worsened features of colitis. Colitis-associated phosphorylation of p38, ERK1/2 and Akt was reduced by Ang 1–7 treatment. Conclusion Our results indicate important anti-inflammatory actions of Ang 1–7 in the pathogenesis of IBD, which may provide a future therapeutic strategy to control the disease progression.

Enaminones and norepinephrine employ convergent mechanisms to depress excitatory synaptic transmission in the rat nucleus accumbens in vitro.

We recently reported that anticonvulsant anilino enaminones depress excitatory postsynaptic currents (EPSCs) in the nucleus accumbens (NAc) indirectly via γ‐aminobutyric acid (GABA) acting on GABAB receptors [ S.B. Kombian et al. (2005)Br. J. Pharmacol., 145, 945–953]. Norepinephrine (NE) and dopamine (DA), both known to be involved in seizure disorders, also depress EPSCs in this nucleus. The current study explored a possible interaction between enaminones and adrenergic and/or dopaminergic mechanisms that may contribute to their synaptic depression and anticonvulsant effect. Using whole‐cell recording in rat forebrain slices containing the NAc, we show that NE‐induced, but not DA‐induced, EPSC depression occludes E139‐induced EPSC depressant effect. UK14,304, a selective α2 receptor agonist, mimicked the synaptic effect of NE and also occluded E139 effects. Phentolamine, a non‐selective α‐adrenergic antagonist that blocked NE‐induced EPSC depression, also blocked the E139‐induced EPSC depression. Furthermore, yohimbine, an α2‐adrenoceptor antagonist, also blocked the E139‐induced EPSC depression, while prazosin, a selective α1‐adrenergic antagonist, and propranolol, a non‐selective β‐adrenoceptor antagonist, did not block the E139 effect. Similar to the E139‐induced EPSC depression, the NE‐induced EPSC depression was also blocked by the GABAB receptor antagonist, CGP55845. By contrast, however, neither SCH23390 nor sulpiride, D1‐like and D2‐like DA receptor antagonists, respectively, blocked the E139‐induced synaptic depression. These results suggest that NE and E139, but not DA, employ a similar mechanism to depress EPSCs in the NAc, and support the hypothesis that E139, like NE, may act on α2‐adrenoceptors to cause the release of GABA, which then mediates synaptic depression via GABAB receptors.

Synthesis, neuronal activity and mechanisms of action of halogenated enaminones

Due to the excellent anticonvulsant activity of previously synthesized halogenated enaminones, more disubstituted analogs were synthesized and evaluated in vitro. The new enaminones either had no effect, depressed, or enhanced population spike (PS) amplitude in the rat hippocampus in a concentration-dependent manner. Structure-activity relationship (SAR) analysis indicated that compounds 21 and 25 (with dibromo substituents) were equipotent, and more potent than compound 2 (with dichloro substituents), with compound 25 being the most efficacious of all tested compounds. Both diiodo derivatives 30 and 31 tested produced no significant effect on PS. For PS depression, phenyl substitution on the cyclohexenone ring produced the most efficacious compound 25. PS depressing analogues also depressed evoked excitatory postsynaptic current (EPSC) and action potential firing frequency. Removal of phenyl or methyl group from position 6 on the cyclohexenone ring of enaminone esters produced compound 28 which exhibited pro-convulsant effects. There was no direct correlation between C log P values and anticonvulsant activity of the halogenated enaminones. The mechanisms of anticonvulsant activity were the indirect suppression of excitatory synaptic transmission by enhancing extracellular GABA, and the direct suppression of action potential firing of the neurons.

Cholecystokinin inhibits evoked inhibitory postsynaptic currents in the rat nucleus accumbens indirectly through γ-aminobutyric acid and γ-aminobutyric acid type B receptors

We recently reported that cholecystokinin (CCK) excited nucleus accumbens (NAc) cells and depressed excitatory synaptic transmission indirectly through γ‐aminobutyric acid (GABA), acting on presynaptic GABAB receptors (Kombian et al. [ 2004 ] J. Physiol. 555:71–84). The present study tested the hypothesis that CCK modulates inhibitory synaptic transmission in the NAc. Using in vitro forebrain slices containing the NAc and whole‐cell patch recording, we examined the effects of CCK on evoked inhibitory postsynaptic currents (IPSCs) recorded at a holding potential of −80 mV throughout CCK‐8S caused a reversible inward current accompanied by a concentration‐dependent decrease in evoked IPSC amplitude. Maximum IPSC depression was ∼25% at 10 μM, with an estimated EC50 of 0.1 μM. At 1 μM, CCK‐8S induced an inward current of 28.3 ± 4.8 pA (n = 6) accompanied by an IPSC depression of −18.8% ± 1.6% (n = 6). This CCK‐induced IPSC depression was blocked by pretreatment with proglumide (100 μM; −3.7% ± 6.9%; n = 4) and by LY225910 (100 nM), a selective CCKB receptor antagonist (4.4% ± 2.6%; n = 4). It was not blocked by SCH23390 (10 μM; −23.5% ± 1.3%; P < 0.05; n = 7) or sulpiride (10 μM; −21.8% ± 5.1%; P < 0.05; n = 4), dopamine receptor antagonists. By contrast, it was blocked by CGP55845 (1 μM; −0.4% ± 3.4%; n = 5) a potent GABAB receptor antagonist, and by forskolin (50 μM; 9.9% ± 5.2%; n = 4), an adenylyl cyclase activator, and H‐89 (1 μM; 6.9% ± 3.9%; n = 4), a protein kinase A (PKA) inhibitor. These results indicate that CCK acts on CCKB receptors to increase extracellular levels of GABA, which then acts on GABAB receptors to decrease IPSC amplitude.

Substance P and cocaine employ convergent mechanisms to depress excitatory synaptic transmission in the rat nucleus accumbens in vitro

Substance P (SP) has been reported to produce effects on excitatory synaptic transmission in the nucleus accumbens (NAc) that are similar to those induced by cocaine. To address the question of whether SP serves as an endogenous mediator producing cocaine‐like effects that are known to be D1‐receptor‐mediated, we tested the hypothesis that the effects of SP and cocaine on excitatory postsynaptic currents (EPSCs) in the NAc occlude one another. We report here that SP and SP5–11 actions occlude the effect of cocaine and vice versa. SP, SP5–11 and cocaine all depressed evoked, non‐N‐methyl‐d‐aspartate (NMDA) receptor‐mediated synaptic currents in a concentration‐dependent manner, with EC50 values of 0.12, 0.17 and 8.3 μm, respectively. Although cocaine was the least potent, it was most efficacious. SP, SP5–11 and cocaine all suppressed isolated NMDA receptor‐mediated evoked EPSCs. SP5–11 (1 μm)‐induced EPSC depression was blocked by the neurokinin‐1 antagonist L732138 and by the D1‐like receptor antagonist SCH23390. Pretreatment of slices with cocaine (30 μm) depressed the EPSC by 39.1% ± 4.8%. Application of SP or SP5–11 (1 μm) at the peak of the cocaine depressive effect on the EPSC did not produce any additional diminution of the response (5.7% ± 2.8%). In the reverse experiments, in which either SP or SP5–11 was applied first, subsequent application of cocaine at the peak of the peptide’s effect (30.3% ± 2.3%) produced a further but smaller depression (15.5% ± 3.6%) of the remaining EPSC. These data indicate that cocaine and SP produce similar effects on excitatory synaptic transmission in the NAc, and that their actions occlude one another. This suggests that SP may act like cocaine in its absence, and may be an endogenous trigger for the reward and behaviors associated with cocaine.

Evaluation of Anticonvulsant Actions of Dibromophenyl Enaminones Using In Vitro and In Vivo Seizure Models

Epilepsy and other seizure disorders are not adequately managed with currently available drugs. We recently synthesized a series of dibromophenyl enaminones and demonstrated that AK6 and E249 were equipotent to previous analogs but more efficacious in suppressing neuronal excitation. Here we examined the actions of these lead compounds on in vitro and in vivo seizure models. In vitro seizures were induced in the hippocampal slice chemically (zero Mg2+ buffer and picrotoxin) and electrically using patterned high frequency stimulation (HFS) of afferents. In vivo seizures were induced in rats using the 6 Hz and the maximal electroshock models. AK6 (10 µM) and E249 (10 µM) depressed the amplitude of population spikes recorded in area CA1 of the hippocampus by −50.5±4.3% and −40.1±3.1% respectively, with partial recovery after washout. In the zero Mg2+ model, AK6 (10 µM) depressed multiple population spiking (mPS) by −59.3±6.9% and spontaneous bursts (SBs) by −65.9±7.2% and in the picrotoxin-model by −43.3±7.2% and −50.0±8.3%, respectively. Likewise, E249 (10 µM) depressed the zero-Mg2+-induced mPS by −48.8±9.5% and SBs by −55.8±15.5%, and in the picrotoxin model by −37.1±5.5% and −56.5±11.4%, respectively. They both suppressed post-HFS induced afterdischarges and SBs. AK6 and E249 dose-dependently protected rats in maximal electroshock and 6 Hz models of in vivo seizures after 30 min pretreatment. Their level of protection in both models was similar to that obtained with phenytoin Finally, while AK6 had no effect on locomotion in rats, phenytoin significantly decreased locomotion. AK6 and E249, suppressed in vitro and in vivo seizures to a similar extent. Their in vivo activities are comparable with but not superior to phenytoin. The most efficacious, AK6 produced no locomotor suppression while phenytoin did. Thus, AK6 and E249 may be excellent candidates for further investigation as potential agents for the treatment of epilepsy syndromes with possibly less CNS side effects.

Anti-Inflammatory Properties of the Enaminone E121 in the Dextran Sulfate Sodium (DSS) Colitis Model.

Background Enaminones are synthetic compounds with an established role in the prevention of various forms of seizures. Recent evidence suggests potent anti-tussive, bronchodilation and anti-inflammatory properties. Pre-treatment with particularly E121 compound resulted in a decrease in leukocyte recruitment in the ovalbumin induced-model of asthma, immune cell proliferation and cytokine release in vitro. We hypothesize that E121 might serve as a therapeutic potential in intestinal inflammation through modulating immune cell functions. Methods Colitis was induced by daily dextran sulfate sodium (DSS) administration for 5 days, and its severity was determined by gross and histological assessments. The plasma level of various cytokines was measured using flow cytometry-based assay. The colonic expression/ phosphorylation level of various molecules was determined by immunofluorescence and western blotting. The effects of E121 treatment on in vitro neutrophil chemotaxis (under-agarose assay), superoxide release (luminol oxidation assay) and apoptosis (annexin V/7AAD) were also determined. Results DSS-induced colitis in mice was significantly reduced by daily E121 treatment (30–100 mg/kg) at gross and histological levels. This effect was due to modulated plasma levels of interleukin (IL-2) and colonic expression levels of various signaling molecules and proteins involved in apoptosis. In vitro neutrophil survival, chemotaxis, and superoxide release were also reduced by E121 treatment. Conclusion Our results indicate important anti-inflammatory actions of E121 in the pathogenesis of IBD.