Shyam Sunder Chatterjee

Hyperforin enhances the extracellular concentrations of catecholamines, serotonin and glutamate in the rat locus coeruleus

Hyperforin is the main antidepressant component of hypericum perforatum (St. Johns Wort). Using the push-pull superfusion technique we tested whether hyperforin influences extracellular concentrations of neurotransmitters in the rat locus coeruleus. Hyperforin (10 mg/kg, i.p.) not only enhanced the extracellular levels of the monoamines dopamine, noradrenaline and serotonin, but also that of the excitatory amino acid glutamate. The levels of the main serotonin metabolite 5-hydroxyindolacetic acid, as well as those of the amino acids GABA, taurine, aspartate, serine and arginine, were not influenced. Together with in vitro studies, our findings suggest that the antidepressant property of hyperforin is due to enhanced concentrations of monoamines and glutamate in the synaptic cleft, probably as a consequence of uptake inhibition.

Phospholipid breakdown and choline release under hypoxic conditions: inhibition by bilobalide, a constituent of Ginkgo biloba.

A marked increase of choline release from rat hippocampal slices was observed when the slices were superfused with oxygen-free buffer, indicating hypoxia-induced hydrolysis of choline-containing phospholipids. This increase of choline release was suppressed by bilobalide, an ingredient of Ginkgo biloba, but not by a mixture of ginkgolides. The EC50 value for bilobalide was 0.38 microM. In ex vivo experiments, bilobalide also inhibited hypoxia-induced choline release when given p.o. in doses of 2-20 mg/kg 1 h prior to slice preparation. The half-maximum effect was observed with 6 mg/kg bilobalide. A similar effect was noted after p.o. administration of 200 mg/kg EGb 761, a ginkgo extract containing approximately 3% of bilobalide. We conclude that ginkgo extracts can suppress hypoxia-induced membrane breakdown in the brain, and that bilobalide is the active constituent for this effect.

Microstructured scaffolds for liver tissue cultures of high cell density: Morphological and biochemical characterization of tissue aggregates

Very high cell densities and optimal vascularization characterize among others organs and tissues in vivo. In order to study organ‐specific functions in vitro or to make use of them in medical devices/treatments in the future, this natural architecture should be rebuilt. An important aspect in this context is the appropriate ratio of medium to cell volume being so far not optimally reestablished in most of the currently available in vitro systems. To improve such culture conditions, we constructed a microstructure to culture hepatocytes and (without any addition of extracellular matrix material) characterized liver tissue in the form of evenly sized aggregates. The liver‐specific differentiation status of such aggregates was monitored by their ability to perform CYP450 dependent xenobiotic metabolism along with the measurement of albumin secretion. Freshly isolated adult rat hepatocytes show an initial loss of total CYP450 content and of associated activities (mixed function oxidases). However, in the aggregate system, this level did not decrease further but remained stable or even increased throughout the culture period of 10–13 days. The CYP450 dependent metabolism of the hepatocytes is able to respond to classic inducing agents. The described culture efficiently supports liver‐specific functions of adult rat hepatocytes and seems to be suited not only for use in an extracorporeal liver device but also for the formation of evenly sized small aggregates to be of use in transplantation of differentiated liver tissue. Moreover, after design variations, the microstructure can be applied for functional analysis of metabolically active hepatocytes as well as for toxicological and pharmacological validation.

BN52021, a platelet activating factor antagonist, is a selective blocker of glycine-gated chloride channel

We have found that the platelet activating factor antagonist (BN52021) is an effective blocker of the glycine (Gly) receptor-mediated responses in the hippocampal pyramidal neurons of rat. Using the whole-cell voltage clamp and concentration clamp recording techniques, we investigated the mechanism underlying the inhibitory action of this terpenoid on the glycine-induced chloride current. BN52021 selectively and reversibly inhibits glycine current in a non-competitive and voltage-dependent fashion. The antagonistic effect of this substance is more pronounced at positive membrane potentials. At holding potential -70mV and in the presence of 200 microM glycine IC50 value for the blocking action of BN52021 was 270+/-10nM. Repetitive applications of BN52021 reveal the use-dependence of its blocking action. When co-applied with strychnine (STR), a competitive glycine receptor antagonist, BN52021 does not alter the IC50 value for strychnine. The inhibitory effect of BN52021 on gamma-aminobutyric acid (GABA) current is at least 25 times less potent than the effect on glycine current. This substance fails to affect AMPA and NMDA responses. It may be concluded that BN52021 inhibits glycine-gated Cl- channels by interacting with the pore region and does not compete for the strychnine-binding centre.

Stimulation of hippocampal acetylcholine release by hyperforin, a constituent of St. John’s Wort

Extracts of the medicinal plant St. Johns Wort (Hypericum perforatum) are widely used in the therapy of affective disorders and have been reported to exert antidepressant, anxiolytic, and cognitive effects in experimental and clinical studies. We here report that hyperforin, the major active constituent of the extract, increases the release of acetylcholine from rat hippocampus in vivo as determined by microdialysis. Hippocampal acetylcholine levels were increased by 50-100% following the systemic administration of pure hyperforin at doses of 1 and 10 mg/kg. The effect was almost completely suppressed by local perfusion with calcium-free buffer or with tetrodotoxin (1 microM). We conclude that hyperforin releases hippocampal acetylcholine by an indirect mechanism of action which is calcium-dependent and requires intact neuronal communication and cell firing. Our findings suggest therapeutic efficacy of St. Johns Wort extracts in central cholinergic dysfunction.

KA-672 inhibits rat brain acetylcholinesterase in vitro but not in vivo

KA-672, a lipophilic benzopyranone derivative which is currently under development as a cognitive enhancer and antidementia drug, has previously been shown to have facilitatory effects on learning and memory in rats at doses of 0.1-1 mg/kg. We now report that KA-672 inhibited the activity of acetylcholinesterase (AChE), measured in vitro in rat brain cortical homogenate, with an IC50 value of 0.36 microM indicating that KA-672 may improve cognitive functions as a consequence of AChE inhibition. However, when we employed the microdialysis procedure to monitor acetylcholine (ACh) release from rat hippocampus, no effect of KA-672 (0.1-10 mg/kg) was found, indicating a lack of inhibition of brain AChE under in vivo-conditions. [14C]-labelled KA-672 was found to easily penetrate the blood-brain barrier, and an apparent concentration of 0.22 nmol/g brain (equivalent to 0.39 microM tissue concentration) was calculated following an i.p. injection of 1 mg/kg KA-672. However, no labelled substance could be detected in hippocampal microdialysates or in cerebrospinal fluid (CSF) taken from the cisterna magna, indicating that the concentration of KA-672 in brain extracellular fluid must have been below 0.01 microM. We conclude that KA-672 is a potent AChE inhibitor, an activity which, however, does not contribute to its behavioural effects in vivo because the lipophilic drug does not reach sufficient concentrations in the extracellular fluid, apparently due to cellular sequestration.

Inhibition of hippocampal LTP by ginkgolide B is mediated by its blocking action on PAF rather than glycine receptors

Platelet-activating factor (PAF), a biologically active lipid (1-O-alkyl-2-acetyl-sn-glycero-3-phosphoholine), is identified in different regions of brain, including hippocampus. Specific PAF-activated receptors (PAFRs) are expressed in corresponding brain areas. PAF has been proposed to be a retrograde messenger of long-term potentiation (LTP): the antagonist of PAFRs, ginkgolide B (or BN52021) prevents induction of LTP. Recently it has been found that ginkgolide B is also an efficient blocker of the glycine receptor (GlyR) operated chloride channels (IC(50)=270+/-10 nM in hippocampal pyramidal neurons). The question is as follows: is the alteration of LTP by BN52021 due to the PAF antagonism or to the inhibition of glycine-gated chloride channels? We have studied the effects of ginkgolides B and J on LTP induced in the CA1 area of rat hippocampus. Ginkgolide J which is the weakest blocker of PAFR (IC(50)=54 microM, as compared to IC(50)=2.5 microM for ginkgolide B) inhibits GlyR-operated channels with IC(50)=2.0 microM. This assures a convenient concentration window which allows to inhibit GlyR-operated channels without affecting PAFRs. An amount of 5 microM of ginkgolide J did not prevent the induction of LTP, while ginkgolide B (5 microM) completely inhibited this phenomenon. The effect of ginkgolide B on LTP did not alter considerably if GlyRs were blocked by strychnine (2 microM). Strychnine itself had no significant effect on the induction of LTP. Both ginkgolides and strychnine significantly facilitated short-term potentiation (STP). Our data support a hypothesis according to which ginkgolides affect LTP by inhibiting PAFRs.

Ginkgolide B preferentially blocks chloride channels formed by heteromeric glycine receptors in hippocampal pyramidal neurons of rat

It has been found recently that the platelet activating factor antagonist ginkgolide B is a selective use-dependent blocker of glycine-gated chloride channels. GABAA receptor antagonist picrotoxin is known to block alpha homomeric glycine (Gly) receptors, being less effective for heteromeric alpha1/beta glycine receptors. Studying pyramidal hippocampal neurons of rat, we have confirmed that the effect of picrotoxin depends on the age of the animals. Its blocking ability was characterised by IC50=140+/-12 microM and IC50=354+/-43 microM for 7 and 14 days old rats, respectively, indicating at a possibly increased contribution of heteromeric receptors with animals age. We have revealed that the blocking action of ginkgolide B is subjected to a more drastic change in the same range of ages: the IC50 value is decreased from 1.6+/-0.2 microM for 7 days old rats to 0.27+/-0.01 microM for 14 days old rats. When measured on the background of ginkgolide B (1 microM), IC50 for picrotoxin was 92+/-16 microM. Taken together, these findings indicate that ginkgolide B has higher affinity to heteromeric Gly receptor-gated channels than to the homomeric ones.

Influence of the Ginkgo extract EGb 761 on rat liver cytochrome P450 and steroid metabolism and excretion in rats and man

Extracts from leaves of Ginkgo biloba L. are among the most used herbal medicinal products worldwide. Based on in‐vitro tests and studies in rats, concern has been expressed that intake of Ginkgo extracts may affect hepatic metabolism of xenobiotics and cause drug interactions, although no evidence for modulation of cytochrome P450 (CYP450) enzyme activity was obtained in human trials. Because of these contradictory findings, we investigated the effects of the standardised extract EGb 761 on hepatic CYP450 in rats. EGb 761 (100 mg kg−1 daily, p.o., for 4 days) strongly increased liver CYP450 content and altered the ex‐vivo biotransformation of androstendione, as well as metabolism of endogenous steroids. However, in human subjects no effect on the urinary steroid profile was observed after intake of EGb 761 for 28 days (240 mg daily). These results indicate that the effects of EGb 761 on drug metabolising enzymes are specific for rats and may not be extrapolated to man.

Acute effect of KA-672, a putative cognitive enhancer, on neurotransmitter receptor binding in mouse brain

7-Methoxy-6-[3-[4-(2-methoxyphenyl)piperazin-1-yl]propoxy]-3,4-dim ethyl-2H-1-benzopyran-2-one hydro-chloride (KA-672), structurally related to naturally occurring coumarins, has been described as a potential drug for enhancing cognitive functions. However, a detailed characterization of the pharmacological profile of KA-672 in vivo is still lacking. Quantitative neurotransmitter receptor autoradiography was used as a tool to screen for KA-672-induced changes in a number of transmitter receptors including cholinergic, noradrenergic, glutamatergic, GABAergic, and serotonergic subtypes throughout the brain. Two hours following treatment of mice with 1 mg/kg KA-672 per os, slight increases of nicotinic and M1-muscarinic cholinergic receptor binding, of alpha2-and beta-adrenoceptor as well as 5-HT1A receptors in various cerebral cortical regions were observed, whereas 5-HT2A binding sites were strikingly increased throughout the brain following KA-672 treatment. In contrast, (+/-)-alphaamino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor binding was significantly decreased in some cortical regions after drug treatment. No effects of KA-672 treatment on N-methyl-D-aspartate, kainate, GABA(A) and benzodiazepine receptor as well as M2-muscarinic cholinergic and high-affinity choline uptake binding were observed. As interactions between the cholinergic, noradrenergic and serotonergic neurotransmission have been stressed to play important roles in realizing learning and memory events, the cognition-enhancing effects of KA-672 may be due to this complex in vivo pharmacological profile of KA-672.