Daniel A. García

Allosteric positive interaction of thymol with the GABAA receptor in primary cultures of mouse cortical neurons

Thymol is a naturally occurring phenolic monoterpene known for its anti-microbial and anti-oxidant properties. It is used in dental practice and in anaesthetic halothane preparations. Recent studies have reported enhanced GABA(A) receptor-operated chloride channel activity and increased binding affinity of [(3)H]flunitrazepam in the presence of thymol. In the present work, we more closely examined the pharmacological action of thymol on the native GABA(A) receptor by using primary cultures of cortical neurons. Thymol enhanced GABA-induced (5 microM) chloride influx at concentrations lower than those exhibiting direct activity in the absence of GABA (EC(50) = 12 microM and 135 microM, respectively). This direct effect was inhibited by competitive and non-competitive GABA(A) receptor antagonists. Thymol increased [(3)H]flunitrazepam binding (EC(50) = 131 microM) and showed a tendency to increase [(3)H]muscimol binding. These results confirm that thymol is a positive allosteric modulator of the GABA(A) receptor. The thymol structural analogues menthol and cymene, which lack an aromatic ring or a hydroxyl group, did not affect [(3)H]flunitrazepam binding. Using a pharmacophoric model that includes a hydrogen bond donor group as well as an aromatic ring with two aliphatic substituents, we propose to demonstrate the molecular essential features of these compounds to interact with GABA(A) receptors. Thymol (0-1 mM) did not affect cellular viability.

Studies with neuronal cells: From basic studies of mechanisms of neurotoxicity to the prediction of chemical toxicity

Neurotoxicology considers that chemicals perturb neurological functions by interfering with the structure or function of neural pathways, circuits and systems. Using in vitro methods for neurotoxicity studies should include evaluation of specific targets for the functionalism of the nervous system and general cellular targets. In this review we present the neuronal characteristics of primary cultures of cortical neurons and of cerebellar granule cells and their use in neurotoxicity studies. Primary cultures of cortical neurons are constituted by around 40% of GABAergic neurons, whereas primary cultures of cerebellar granule cells are mainly constituted by glutamatergic neurons. Both cultures express functional GABAA and ionotropic glutamate receptors. We present neurotoxicity studies performed in these cell cultures, where specific neural targets related to GABA and glutamate neurotransmission are evaluated. The effects of convulsant polychlorocycloalkane pesticides on the GABAA, glycine and NMDA receptors points to the GABAA receptor as the neural target that accounts for their in vivo acute toxicity, whereas NMDA disturbance might be relevant for long-term toxicity. Several compounds from a list of reference compounds, whose severe human poisoning result in convulsions, inhibited the GABAA receptor. We also present cell proteomic studies showing that the neurotoxic contaminant methylmercury affect mitochondrial proteins. We conclude that the in vitro assays that have been developed can be useful for their inclusion in an in vitro test battery to predict human toxicity.

The light-harvesting complexes of a thermophilic purple sulfur photosynthetic bacterium Chromatium tepidum

Abstract Several biophysical properties (absorption, fluorescence, linear dichroism) are reported for the chromatophore membranes of the thermophilic purple sulfur bacterium, Chromatium tepidum. Like the mesophilic strain Chromatium vinosum, two types of light-harvesting complex are present: one absorbs at 800 nm and 855 nm; the second complex is equivalent to the B890 complex of C. vinosum, but absorbs at 918 nm, i.e., 30 nm higher. This is the highest absorption band observed so far for a light-harvesting complex containing bacteriochlorophyll a. In spite of the small overlap between the fluorescence and absorption bands of the two light-harvesting complexes, specially at low temperature, an efficient energy transfer occurs from the high-energy (B800–855) to the low-energy (B920) complexes. The B800–855 complexes have been isolated from the whole membrane by lauryldimethylamine N-oxide treatment, whereas only a partial purification was achieved for the B920 complexes.

Lipophilicity of some GABAergic phenols and related compounds determined by HPLC and partition coefficients in different systems

Some phenolic compounds, like propofol and thymol, have been shown to act on the GABA(A) receptor. Taking into account the hydrophobicity of these compounds, their interaction with the membrane surrounding the receptor and consequent non-specific effect on receptor modulation cannot be neglected. In the present work, we determined and correlated several lipophilic parameters for both GABAergic agents and three other related phenolic compounds (eugenol, carvacrol and chlorothymol), including logP(o/w), retention data in high performance liquid chromatography (HPLC) by using C18 and immobilized artificial membrane (IAM) columns at different temperatures, and partition coefficients determined in phospholipid liposomes. The correlation results demonstrated the high capacity of the compounds assayed to interact with phospholipid membrane phases, which can be predicted by simple model systems as logP(o/w) or HPLC. The values obtained by HPLC using a fast screening IAM column were the quantitatively closest to the partition coefficients determined in liposome systems, due to the capacity of this column to permit the establishment of molecular interactions like those found in phospholipid membranes. Finally, the fact that all the compounds studied are able to interact with membranes would suggest the participation of some alteration of the GABA(A) receptor lipid environment as part of the receptor modulation exerted by phenolic compounds.

T-maze behaviour in domestic chicks: a search for underlying variables

We investigated whether contrasting T-maze behaviour shown by domestic chicks, Gallus gallus domesticus, of a broiler strain reflected underlying differences in their general activity levels, fearfulness or sociality. The time taken by 2-day-old chicks to traverse a T maze and thereby regain visual contact with their companions was measured. Chicks were categorized according to whether they completed this task quickly (HP, high performance, <25 s) or slowly (LP, low performance, >75 s) and then housed in same-category groups, each of eight chicks. In experiment 1, we compared the numbers of HP and LP chicks showing certain home cage behaviours (ambulation, standing, resting, maintenance, pecking, preening). No significant differences were found. In experiment 2, we compared the behaviour of HP and LP chicks in two tests of sociality (home cage proximity, runway) and in two tests of fear (emergence, tonic immobility). The HP chicks stayed closer together in the home cage than did their LP counterparts and they spent significantly longer near a goal box containing conspecifics in the runway than did LP ones. Conversely, there were no significant differences between HP and LP chicks in their latency to emerge from a sheltered area into an exposed and, hence, potentially frightening one or in their tonic immobility fear reactions. These findings suggest that contrasting T-maze performance was unlikely to have reflected differences in underlying activity levels or in fearfulness. Conversely, individual variation in underlying sociality was probably an influential variable. Copyright 1999 The Association for the Study of Animal Behaviour.

Localization of flunitrazepam in artificial membranes. A spectrophotometric study about the effect the polarity of the medium exerts on flunitrazepam acid-base equilibrium

In the present paper we tried to test the hypothesis that nonspecific flunitrazepam-membrane interactions are consistent with drug molecules accommodated between lipid molecules, becoming an integral part of the bilayer. We developed a spectrophotometric method to determine FNTZH+ equilibrium dissociation constant and applied it to the study of the acid-base equilibria of this drug in homogeneous media of different polarity. In these conditions, pK decreased with the decrement in the dielectric constant (D) of the media. These results, analyzed under the light of the theory developed by Fernandez and Fromherz (1977; J. Phys. Chem. 81, 1755-1761) let us infer that flunitrazepam is localized a region with D = 60. This D value is lower that Dwater = 78 and higher than D of hydrocarbon chains zone (D = 2-5) and would correspond to D of the region of polar groups. This result is compatible with the hypothesis.

Tagetone modulates the coupling of flunitrazepam and GABA binding sites at GABAA receptor from chick brain membranes.

The effects of tagetone on flunitrazepam (FNTZ) binding to synaptosomal membranes from chick brains in the presence and absence of allosteric modulations induced by gamma-aminobutyric acid (GABA) were investigated. Tagetone, at 50 micrograms/ml (final concentration), decreased the binding affinity of [3H]FNTZ to synaptosomal membranes form chick brain (Kd = 3.34 +/- 0.36 nM without tagetone and Kd,t = 5.86 +/- 0.86 nM with tagetone; p < 0.05, two tailed Students t-test) without affecting maximal binding (Bmax = 488 +/- 24 fmoles/mg protein, and Bmax,t = 500 +/- 25 fmoles/mg protein in the absence and in the presence of tagetone respectively). The potency of GABA to stimulate [3H]FNTZ binding increased in the presence of tagetone (EC50 values were 2.78 and 1.12 microM with and without tagetone respectively). GABA was able to decrease merocyanine delta A570-610 values in a concentration dependent manner; half maximal effect was attained at a GABA concentration of 34 +/- 13 microM. Tagetone, at a concentration of 50 micrograms/ml and in the presence of GABA 30 microM or 60 microM, enhanced the ability of GABA alone on decreasing delta A570-610. Tagetone alone did not change delta A570-610 values. FNTZ, a well known GABA modulator, could also potentiate the effect of GABA. Theoretical calculations indicate that the effects on merocyanine delta A570-610 value are mainly exerted at the membrane potential level (delta psi m). The present results strongly suggest that tagetone affected the function of GABAA receptor in a complex way: on the one hand it impaired FNTZ binding: on the other hand tagetone improved both the coupling between FNTZ and GABA binding sites and it enhanced GABA-induced chloride permeability. Changes in the geometrical and electrostatic properties of the self-organized membrane structure may account for these effects of tagetone.

Effects of propofol and other GABAergic phenols on membrane molecular organization

GABA(A) receptor is the main inhibitory receptor of the central nervous system. The phenols propofol and thymol have been shown to act on this receptor. GABA(A) is an intrinsic protein, the activity of which may be affected by physical changes in the membrane. Taking into account the lipophilicity of phenols, their interaction with the membrane and a consequent non-specific receptor modulation cannot be discarded. By using Langmuir films, we analyze the comparative effects on the molecular properties of the membrane exerted by propofol, thymol and other related compounds, the activities of which on the GABA(A) are under investigation in our laboratory. All the compounds were able to expand phospholipid films, by their incorporation into the monolayer being favored by less-packed structures. Nonetheless, they were able to be incorporated at lateral pressures above the equilibrium pressure estimated for a natural membrane. Epifluorescence images revealed their presence between phospholipid molecules, probably at the head-group region. Hence, all results indicated that the phenols studied were clearly able to interact with membranes, suggesting that their anesthetic activity could be the combined result of their interaction with specific receptor proteins and with their surrounding lipid molecules modulating the supramolecular organization of the receptor environment.

Structure of Chloroflexus aurantiacus reaction center: Photoselection at low temperature

Abstract Photoselection experiments have been performed on isolated Chloroflexus aurantiacus reaction centers at 20 K. Our data show that the average angle between the ground state BPh Qy transitions and the 890 nm transition is approx. 50°. Only two BPh Qy transitions are affected by the charge separation. These two transitions are perpendicular to the long-wavelength band of the primary donor. The ground state of the 813 nm transition makes an angle of 35° with the dimer absorption band. The polarization ratio of the light-induced absorption decrease at 815 nm is not consistent with that decrease being due solely to an electrochromic bandshift of the 813 nm transition.

Supramolecular events modulate flunitrazepam partitioning into natural and model membranes

In the present paper we investigate the effects of chemical and physical variables which modulate supramolecular membrane organization on the partitioning of FNTZ into natural and model membranes. In dpPC the value of PdpPC-B increased almost discontinuously at a temperature close to the gel-liquid crystalline phase transition temperature of dpPC (41.5°C). In natural membranes, Pmb remained constant upto 37°C and increased significatively at higher temperatures. However, the ascending slope was less sharp than in liposomes, reflecting a lower cooperativity of the change due to the higher complexity of the natural membrane compared with that of the pure lipid. As a function of pH, P values increased slightly in dpPC, increased markedly in dpPE, decreased in dpPS and, in dpPA, P decreased up to pH 8 and, at higher pH values, it tended to increase again. The variation in P as a function of pH resulted from the balance between two opposite tendencies: (1) changes in the viscosity of phospholipid bilayers as a function of pH due to modification of the surface net charge leading to changes the packing of the lipid molecules, and (2) the reduction of interface hydration induced by the decreasing pH which enhances its hydrophobicity. The former membrane property is favored up to pH 7. Partitioning of FNTZ is favored in less hydrated and less ordered interfaces. At pH < 3, FNTZ stablishes with the lipid-interface attractive (dpPS and dpPA) or repulsive (dpPE) electrostatic interactions, which can be screened by the ions arising from salt dissociation over a certain concentration level. The increase in the proportion of cholesterol in dpPC-cholesterol mixed liposomes induces a decrease in the value of P. This result may be explained by the assumption that possible vacancies which might incorporate FNTZ are already filled with cholesterol, thus inhibiting further FNTZ incorporation. The membrane-buffer partition coefficient of FNTZ is strongly influenced by the physical state of the bilayer membrane. Particular interactions may lead to a strong local accumulation of FNTZ within the membrane due to a coupling to lateral density fluctuations.