María A. Perillo

Determination of the membrane-buffer partition coefficient of flunitrazepam, a lipophilic drug.

The partition coefficient (P) of a benzodiazepine, flunitrazepam (FNTZ), was determined in a synaptosomal membrane/buffer system. A two component model was used, one of the components reflecting the drug partitioning into the membrane, and the other the amount of drug in the aqueous phase retained by the pellet after the centrifugation. The quantity of [3H]FTNZ measured as nonspecifically bound to the membrane includes both components so, the second one had to be discounted and in order to determined its magnitude a parallel experiment was performed using a non-partitioning hydrophilic drug (gamma-[3H]aminobutyric acid, [3H]GABA). The assay required previous determination of the fraction of the total volume of the incubation system that corresponded to membrane (fm). The fm value was calculated from the density value (delta) determined by a picnometer method. The results obtained were: delta = 1.66 +/- 0.02; fm = (1.6 +/- 0.2) 10(-3); P = 18.5 +/- 0.8. This P value could explain nonspecific effects of BZDs on some functions of the neuronal membrane.

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.

Open-Field Temporal Pattern of Ambulation in Japanese Quail Genetically Selected for Contrasting Adrenocortical Responsiveness to Brief Manual Restraint

Japanese quail selected for a low-stress (LS), rather than a high-stress (HS), plasma corticosterone response to brief restraint have been shown to possess lower fearfulness and a nonspecific reduction in stress responsiveness. Detrended fluctuation analysis provides information on the organization and complexity of temporal patterns of behavior. The present study evaluated the temporal pattern of ambulation of LS and HS quail in an open field that represented a novel environment. Time series of 4,200 data points were collected for each bird by registering the distance ambulated every 0.5 s during a 35-min test period. Consistent with their known reduced fearfulness, the LS quail initiated ambulation significantly sooner (P < 0.02) and tended to ambulate more (P < 0.09) than did their HS counterparts. Detrended fluctuation analyses showed a monofractal series (i.e., a series with similar complexity at different temporal scales) in 72% of the birds. These birds initiated their ambulatory activity in less than 600 s. Among these birds, a lower (P < 0.03) autosimilarity coefficient (alpha) was found in the LS quail than in their HS counterparts (alpha = 0.76 +/- 0.03 and 0.87 +/- 0.03, respectively), suggesting a more complex (less regular) ambulatory pattern in the LS quail. However, when the patterns of ambulation were reexamined by considering only the active period of the time series (i.e., after the birds had initiated their ambulatory activity), monofractal patterns were observed in 97% of the birds, and no differences were found between the lines. Collectively, the results suggest that during the active period of open-field testing, during which fear responses are likely less strong and other motivations are the driving forces of ambulation, the LS and HS lines have similar ambulatory organization.

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.

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.

The essential oil fromTagetes minuta L. modulates the binding of [3H]flunitrazepam to crude membranes from chick brain

The hypothesis that the essential oil fromTagetes minuta L. can interact with biological membranes was investigated by assessing its ability of perturbing the binding of a benzodiazepine [flunitrazepam (FNTZ)] to crude membranes from chick brains. The essential oil fromT. minuta L. inhibited [3H]FNTZ specific binding to chick brain membranes. These values were obtained from the analysis of the saturation curve for the kinetic parameters: dissociation equilibrium constant (Kd)=2.47±0.32 nM, maximal binding (Bmax)=556±5 fmoles/mg protein, and Hill coefficient (n)=1.00±0.07 in the absence, and Kd=6.73±1.4 nM, Bmax=583±69 fmoles/mg protein, and n=1.02±0.08 in the presence of 29 μg/mL of essential oil. The essential oil could self-aggregate with a critical micellar concentration (CMC) of 60 μg/mL. The marked increase in [3H]FNTZ nonspecific binding starting at 60 μg of essence per mL was due to that phenomenon and revealed the ability of self-aggregated structures to interact with membranes. [3H]FNTZ specific binding decrement as a function of essence concentration cannot be ascribed merely to oils micelles ability of trapping the lipophilic radioligand molecules, because the discontinuous behavior that characterizes a monomer-aggregate phase transition was not shown. Oils components might behave as competitive inhibitors or allosteric modulators of FNTZ specific binding. However, their ability to increase FNTZ nonspecific binding at concentrations below oils CMC suggests that this effect may be due to oils partitioning into the lipid bilayer. This latter phenomenon would induce an increment in membrane fluidity and a change on FNTZ binding site toward a lower affinity conformation. Therefore, the essential oil components can interact with brain membranes either as monomers, by partitioning into the lipid bilayer, or as self-aggregated structures, through an adsorption to the membrane surface.

Monoterpenes affect chlorodiazepoxide-micelle interaction through micellar dipole potential modifications.

The ability of several natural terpenes to affect benzodiazepine (BZD)-micelle interaction through the membrane dipolar organization was investigated. The acid-base equilibrium of chlorodiazepoxide (CDX) and the spectroscopic behavior of the electrochromic dye merocyanine were tested in the presence and in the absence of Triton X-100 micelles (used to mimic a membrane environment) containing or not cineole, menthol, geraniol or camphor. CDXs apparent pK increased in the environment of terpene-containing micelles compared with pure Triton X-100 micelles. Decrements in electric potentials (between -111 and -128 mV with respect to pure detergent) were calculated from Boltzmann equation. This result suggested, that in the presence of terpenes, the tendency of CDXH(+) to remain in the membrane phase increased. The dielectric constant (D) of the microenvironment sensed by merocyanine within Triton X-100 micelles, determined from lambda(max,2) of merocyanine monomer, was D=9 and increased in the presence of all the terpenes assayed (D congruent with 11). The decrease in merocyanine partitioning (A(peak1)/A(peak2) increased) also reflected an increment in the negative dipole potential. The present results suggest that terpenes contributed to the whole dipolar arrangement of the micelle with a dipole moment vector which had an intense component oriented parallel to the intrinsic dipole of the Triton X-100 molecules in the micelles. This led to a more negative environment of the interface region where CDX was located, and increased the net polarity of the deepest micelle regions sensed by merocyanine.

Membrane topology modulates β-galactosidase activity against soluble substrates

Abstract The effect of bio-surfaces of contrasting curvature, on the kinetic parameters of ortho-nitrophenyl-β- d -galactopiranoside hydrolysis catalyzed by E. coli β-galactosidase, was investigated. The self-aggregating state and structure of the amphiphiles (Phosphatidylcholine, Lubrol-PX, Triton X-100, DocNa, SDS and CTAB) were inferred from their c.m.c. values and light-scattering measurements. Low curvature phosphatidylcholine or mixed phosphatidylcholine-detergent vesicles increased V max without affecting K M . High curvature micellar structures containing ionic detergents modulated negatively the enzyme activity (decreased or abolished V max and increased K M ). Neither micelles containing non-ionic detergents nor the amphiphiles in a monomeric form, affected enzyme activity. CTAB at a concentration bellow its c.m.c but incorporated into a bilayer, became an activator ( K M decreased respect to the control). Non-enzymatic interfacial hydrolysis of the substrate was discarded. Enzyme–membrane interaction and membrane elasticity, were evaluated using monomolecular layers at the air–water interface. Beyond particular molecular structures, topology affected the direction of the modulatory effects exerted by these amphiphiles on β-galactosidase activity.

Flunitrazepam partitioning into natural membranes increases surface curvature and alters cellular morphology

In recent studies, we showed that flunitrazepam (FNTZ) and other benzodiazepines interact with artificial phospholipid membranes locating at the polar head group region, inducing a membrane expansion, reducing the molecular packing and reorganising molecular dipoles. In the present paper we investigated the possibility that those phenomena could be transduced into changes in the curvature of membranes from natural origin. Hence we studied the effect of FNTZ on cellular morphology using human erythrocyte as a natural assay system. Shape changes of erythrocytes were evaluated by light microscopy and expressed as a morphological index (MI). FNTZ induced echinocytosis in a time-dependent manner with MI values significantly higher than those of control (without drug) or DMSO (vehicle) samples. Lidocaine, a local anesthetic known to induce stomatocytosis by incorporating in the inner monolayer, counterbalanced the concentration-dependent FNTZ crenating effects. FNTZ induced protective effects, compared with control and DMSO, against time-dependent hemolysis. Hypotonic-induced hemolysis, was also lowered by FNTZ in a concentration-dependent manner. Both antihemolytic effects suggested a drug-induced membrane expansion allowing a greater increase in cell volume before lysis. In such a complex system like a cell, curvature changes triggered by drug partitioning towards the plasma membrane, might be an indirect effect exerted through modifications of ionic-gradients or by affecting cytoskeleton-membrane linkage. In spite of that, the curvature changes can be interpreted as a mechanism suitable to relieve the tension generated initially by drug incorporation into the bilayer and may be the resultant of the dynamic interactions of many molecular fluxes leading to satisfy the spontaneous membrane curvature.