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Formation of benzodiazepine-like molecules in rat brain
1172
Third International Symposium
substantia nigra pare reticulata delayed the onset of circling behaviour and ECoG epileptic discharges induced by subsequent injection of TT into the same area. Pretreatment with sodium valproate (60 min) previously or diazepam (30 min before) reduced or prevented behavioural stimulatory effects and decreased the number and frequency of ECoG discharges induced by intraventricular or intrahippocampal TT. Behavioural excitation and lethal effects produced by intrahippocampal TT were abolished by systemic treatment with either competitive (e .g . CGP37849) or non-competitive (e.g. dizolcipine) NMDA receptor complea antagonists. Carbamazepine was also effective. These data support the involvement of excitatory transmission in the behavioural and elcetrocortical effects of TT . Btuce et al. (1985) 1. Physio1368, 343-357 . C~r.~aarsi et al. (1989) Neuroscience 30, 663-670.
Venom toxins of Theraphosidae spiders. G . V. ODELL,' S. A. HunreuxG,' C. Owxev,z E. V. GRISFnN, a l. Mtus and A. Heueear' ('Department of Biochemistry and Physiological Sciences, Oklahoma State University, Stillwater, OK 74078 U.S .A .; 3Shemyakin Institute of Bioorganic Chemistry, Moscow, U.S .S.R.;'Department of Sciences, Oklahoma Baptist University, Shawnee, OK 74081 U.S.A.).
Ttn; sorans content of freeze dried pooled venom samples from three tarantula species varies from 12 to 20% . The venoms are toxic to insects, mice and lizards and are cytotoaic. The use of a volatile buffer permits weight distribution estimations of venom components from a Sephadex G-50 gel filtration column . The venoms contain acylpolyamines, glutamate receptor antagonists, as major venom constitutents. Gel filtration chromatography of Brachypelma emilia, Dugesiella htntzi and Brachypelma smithü venoms show the weight % of acylpolyamines as 36, 63 and 50% respectively . These complexes appear to have structures of indoleacylspermine, with some variations, and are called pseudoargiopinins by Gx~sxnv et al. (1989) . A group of peptide toxins comprise 12 to 30% of the venom and may be classified as muscle necrotic toxins. The sequence of three of these toxins has been established and may be involved in calcium transport. Hyaluronidase is a constituent of the higher molecular weight fraction while the low molecular weight fraction contains the nucleotides ATP, ADP and AMP. The use of reversed phase liquid chromatography resolves multiple components in both the acylpolyamine and peptide toxin fractions. Milligram quantities of toxins are obtained in this manner for further characterization . Progress in this area will be presented. It has been suggested by Ss:rxtvea et a1. (L990) that the indolespermine complexes may serve a function of prey temporary immobilization . Supported by the Oklahoma Agricultural Experiment Station. Gx~sttrx et al. (1989) Toxicon 27, 541-549. Sxnvxere et al. (1990) Toxicon 28, 541-546. Origin ojthe natural benzodiazepines. A. C. P~un~xt (Instituto de Quimica y Fisicoquimica BiolSgicas, Facultad de Farmacia y Hioquimica, Junin 956, Buenos Aires, Argentina) .
Berrzonuz~tr~s are synthetic organic molecules whose anxiolytic, anticonvulsant and sedative actions were discovered serendipitously in 1957. After the pioneering studies made by De Blas and colleagues in 1986, several laboratories have confirmed that mammalian brains contain small amounts of benzodiazepines. Furthermore, traces of benzodiazepines and benzodiazepine-like molecules were found in other drug-free mammalian organs and fluids. Recently, benzodiazepine-like molecules were also detected in many plants including vegetables, grains and flowers. We have found some of these compounds in cow and human milk . In this last fluid we have ban abk to unequivocally identify one of them as diazepam . The explanation of the origin of these organic molecules in higher living beings can be based on two main, and perhaps complementary hypotheses: (a) mammals may be able to synthesize benzodiazepinea; (b) benzodiazepines are synthesized in plants and vegetables either through the agency of their own enzymatic machinery or by the action of contaminating or soil miao-organisms . This process can also operate in the digestive tract of rumiants giving rise to detectable levels oC benzodiazepines in food products . We will present evidence for validity of both hypotheses as well as for the existtnce in nature of other nonbenzodiazepine ligands for the GABA~ receptor. Formation of benzodiazepine-like molecules in rat brain . M. Prv~,' J. M©W~,r A. De Bt.fs; and C. P~Y~' .
('Instituto de Quimica y Fisicoquimica Biolôgicas (UBA-CONICET), Facultad de Fanmacia y Bioquimica,
Third International Symposium
1173
Buenos Aires, Argentina; 2Instituto de Biologia Celular, UBA; 'Division of Molecular Biology and Biochemistry, School of Basic Life Sciences, University of Missouri, U.S .A .). OUR LABORATORY has described the occurrence of benzodiazepines (BZDs) in bovine brain, human plasma and milk. The possible biosynthetic origin of these molecules was investigated in animal tissue and these results are reported here . Rat brain or cortex homogenates or cortical slices were incubated in DMEM medium, 2~ hr at 37°C in a metabolic shaker (5% COaf95% Off. After centrifugation, the supernatant was mixed with a methanolic-acid extract of the pellet and analyzed by HPLC. Radioreceptor analysis and radioimmunoassay with a specific monoclonal antibody detected several active fractions containing substances of mol. wt < 1300 . The estimated global concentration of these molecules was 0.6710.07 ng of Diazepam equivalents/g of wet tissue (n = 13). This activity did not increase by overnight inwbation under non-sterile conditions . Parallel experiments done in the presence of ['H}-Flunitrazepam gave a 67% f7% (n = 4) degree of recovery . Blank values, which were obtained with non-incubated, boiled tissue or incubates carried out at 0°C, contained 0 .13 t 0.02 ng of diazepam equivalents/g wet tissue (n = 7) or no detectable values (n = 6) . No activity was measured in the absence of tissue . These data indicate that under our experimental conditions, BZD-like molecules are formed in rat brain tissue . These are low mol. wt substances, with activity on the BZD receptor and immunoreactivity similar to BZDs . The inhibition ojcalcium release by procaine in skeletal muscle depends on the binding ojthe drug to an intracellular site . G . PIZARRO, S. GONZ~LEZ and BRUM (Dept of Biophysics, School of Medicine, Montevideo, Uruguay).
PROCAtNt: reduces the amplitude of K contractures and calcium transients in whole muscle fibers. In skinned fibers and sarcoplasmic reticulum (SR) vesicles it blocks calcium induced calcium release (CICR) . In order to establish the intra or extracellular nature of the site of action of the drug we studied its effect at different extracellular pHs. Charge movement and calcium transients were recorded simultaneously in single cut fibers of frog skeletal muscle, voltage clamped in a double vaseline gap chamber . One millimole extracelluhir procaine pH = 7 (1% of the drug in its membrane permeable form) reduced the amplitude of the calcium transients elicited by depolarizing pulses by 35% (sem 14%). At pH = 8.5 (24% of the drug uncharged) the reduction was 73% (sem 30%) . Charge movement remained unaffected by the drug. The pH dependence of the effect together with the indemnity of charge movement suggest that the drug traverses the cellular membrane to reach its binding site, located probably at the SR calcium release channel. Procaine could reduce calcium release by a direct action on the RS calcium release channel or by interfering with a presumed CICR mechanism. Supported with funds of Pedeciba and Fundation Manuel Perez. Differences in hippocampal synaptic plasticity in rats with inborn high or low learning ability may be related to different sensitivity ojaspartate receptors . O. A. RA~z (Depto . de Farmacologia, Fac. de C. Quimicas, UNC.
Côrdoba, Argentina) .
RATS with an inborn high (HP) or low (LP) learning capacity were used to study the sensitivity to the blocking effect of OL-2-amino-5-phosphonovaleric acid (APV-10 and 20 pM) on long-term potentiation (LTP) produced in hippotampal slices by a 1 sec tetanus at 200 Hz . The potential evoked by stimulation of the perforant path was recorded from the granulie cell layer of the dentate gyros in 4001an slices perfused with standard Krebs solution or with APV. Ice all slices from HP rats 10 pM and in 85% of the cases at 20 ~M APV did not block LTP generation . In 60% of slices from LP rats APV 10 kM and in all of the cases at 20 PM APV blocked LTP generation . These results are coherent with the hypothesis that the different inborn learning ability of HP and LP rats is related to the different population or sensitivity of N-methyl-n-aspartate (NMDA) receptors. Syntlresis and M.lO-A inhibitory activity ojsome 4-amine phtnethylmninc derivativts . M. REVes,' R. StLVt~RA,2 F.
DArAS~ and B. CASSets' ('Facultad de Ciencias Universidad de Chile, Santiago, Chile; fileurochemistry Division, IIBCE, Montevideo, Uruguay) . T~ 4amine phenethylamine derivatives (APEAD), 4~imethyhunine amphetamine (4DMAA(`)], [4DMAA(-)]; 4~imethylamine phenethylamine (4DMAPEA) and 4-methylamine amphetamine [4-MMAA(+)], were synthesized and assessed for their monoamine oxidase A inhibitory (MAGI-A) effect. The derivatives were synthesized by condensation of appropriate benzaldehyde with nitromethane or nitroethane in presence of buthyl-amine and further reduction with lithium aluminium hydride. The "in vitro" enzymatic inhibition was tested using a mitochondrial suspension from whole rat brain as source of MAO. 5-HT at appropriate concentrations was utilized as selective substrate for MAO-A. The measurements were performed by HPLC with electrochemical detection using 5-HT and 5-HIAA as markers of enzymatic activity.
Third International Symposium
substantia nigra pare reticulata delayed the onset of circling behaviour and ECoG epileptic discharges induced by subsequent injection of TT into the same area. Pretreatment with sodium valproate (60 min) previously or diazepam (30 min before) reduced or prevented behavioural stimulatory effects and decreased the number and frequency of ECoG discharges induced by intraventricular or intrahippocampal TT. Behavioural excitation and lethal effects produced by intrahippocampal TT were abolished by systemic treatment with either competitive (e .g . CGP37849) or non-competitive (e.g. dizolcipine) NMDA receptor complea antagonists. Carbamazepine was also effective. These data support the involvement of excitatory transmission in the behavioural and elcetrocortical effects of TT . Btuce et al. (1985) 1. Physio1368, 343-357 . C~r.~aarsi et al. (1989) Neuroscience 30, 663-670.
Venom toxins of Theraphosidae spiders. G . V. ODELL,' S. A. HunreuxG,' C. Owxev,z E. V. GRISFnN, a l. Mtus and A. Heueear' ('Department of Biochemistry and Physiological Sciences, Oklahoma State University, Stillwater, OK 74078 U.S .A .; 3Shemyakin Institute of Bioorganic Chemistry, Moscow, U.S .S.R.;'Department of Sciences, Oklahoma Baptist University, Shawnee, OK 74081 U.S.A.).
Ttn; sorans content of freeze dried pooled venom samples from three tarantula species varies from 12 to 20% . The venoms are toxic to insects, mice and lizards and are cytotoaic. The use of a volatile buffer permits weight distribution estimations of venom components from a Sephadex G-50 gel filtration column . The venoms contain acylpolyamines, glutamate receptor antagonists, as major venom constitutents. Gel filtration chromatography of Brachypelma emilia, Dugesiella htntzi and Brachypelma smithü venoms show the weight % of acylpolyamines as 36, 63 and 50% respectively . These complexes appear to have structures of indoleacylspermine, with some variations, and are called pseudoargiopinins by Gx~sxnv et al. (1989) . A group of peptide toxins comprise 12 to 30% of the venom and may be classified as muscle necrotic toxins. The sequence of three of these toxins has been established and may be involved in calcium transport. Hyaluronidase is a constituent of the higher molecular weight fraction while the low molecular weight fraction contains the nucleotides ATP, ADP and AMP. The use of reversed phase liquid chromatography resolves multiple components in both the acylpolyamine and peptide toxin fractions. Milligram quantities of toxins are obtained in this manner for further characterization . Progress in this area will be presented. It has been suggested by Ss:rxtvea et a1. (L990) that the indolespermine complexes may serve a function of prey temporary immobilization . Supported by the Oklahoma Agricultural Experiment Station. Gx~sttrx et al. (1989) Toxicon 27, 541-549. Sxnvxere et al. (1990) Toxicon 28, 541-546. Origin ojthe natural benzodiazepines. A. C. P~un~xt (Instituto de Quimica y Fisicoquimica BiolSgicas, Facultad de Farmacia y Hioquimica, Junin 956, Buenos Aires, Argentina) .
Berrzonuz~tr~s are synthetic organic molecules whose anxiolytic, anticonvulsant and sedative actions were discovered serendipitously in 1957. After the pioneering studies made by De Blas and colleagues in 1986, several laboratories have confirmed that mammalian brains contain small amounts of benzodiazepines. Furthermore, traces of benzodiazepines and benzodiazepine-like molecules were found in other drug-free mammalian organs and fluids. Recently, benzodiazepine-like molecules were also detected in many plants including vegetables, grains and flowers. We have found some of these compounds in cow and human milk . In this last fluid we have ban abk to unequivocally identify one of them as diazepam . The explanation of the origin of these organic molecules in higher living beings can be based on two main, and perhaps complementary hypotheses: (a) mammals may be able to synthesize benzodiazepinea; (b) benzodiazepines are synthesized in plants and vegetables either through the agency of their own enzymatic machinery or by the action of contaminating or soil miao-organisms . This process can also operate in the digestive tract of rumiants giving rise to detectable levels oC benzodiazepines in food products . We will present evidence for validity of both hypotheses as well as for the existtnce in nature of other nonbenzodiazepine ligands for the GABA~ receptor. Formation of benzodiazepine-like molecules in rat brain . M. Prv~,' J. M©W~,r A. De Bt.fs; and C. P~Y~' .
('Instituto de Quimica y Fisicoquimica Biolôgicas (UBA-CONICET), Facultad de Fanmacia y Bioquimica,
Third International Symposium
1173
Buenos Aires, Argentina; 2Instituto de Biologia Celular, UBA; 'Division of Molecular Biology and Biochemistry, School of Basic Life Sciences, University of Missouri, U.S .A .). OUR LABORATORY has described the occurrence of benzodiazepines (BZDs) in bovine brain, human plasma and milk. The possible biosynthetic origin of these molecules was investigated in animal tissue and these results are reported here . Rat brain or cortex homogenates or cortical slices were incubated in DMEM medium, 2~ hr at 37°C in a metabolic shaker (5% COaf95% Off. After centrifugation, the supernatant was mixed with a methanolic-acid extract of the pellet and analyzed by HPLC. Radioreceptor analysis and radioimmunoassay with a specific monoclonal antibody detected several active fractions containing substances of mol. wt < 1300 . The estimated global concentration of these molecules was 0.6710.07 ng of Diazepam equivalents/g of wet tissue (n = 13). This activity did not increase by overnight inwbation under non-sterile conditions . Parallel experiments done in the presence of ['H}-Flunitrazepam gave a 67% f7% (n = 4) degree of recovery . Blank values, which were obtained with non-incubated, boiled tissue or incubates carried out at 0°C, contained 0 .13 t 0.02 ng of diazepam equivalents/g wet tissue (n = 7) or no detectable values (n = 6) . No activity was measured in the absence of tissue . These data indicate that under our experimental conditions, BZD-like molecules are formed in rat brain tissue . These are low mol. wt substances, with activity on the BZD receptor and immunoreactivity similar to BZDs . The inhibition ojcalcium release by procaine in skeletal muscle depends on the binding ojthe drug to an intracellular site . G . PIZARRO, S. GONZ~LEZ and BRUM (Dept of Biophysics, School of Medicine, Montevideo, Uruguay).
PROCAtNt: reduces the amplitude of K contractures and calcium transients in whole muscle fibers. In skinned fibers and sarcoplasmic reticulum (SR) vesicles it blocks calcium induced calcium release (CICR) . In order to establish the intra or extracellular nature of the site of action of the drug we studied its effect at different extracellular pHs. Charge movement and calcium transients were recorded simultaneously in single cut fibers of frog skeletal muscle, voltage clamped in a double vaseline gap chamber . One millimole extracelluhir procaine pH = 7 (1% of the drug in its membrane permeable form) reduced the amplitude of the calcium transients elicited by depolarizing pulses by 35% (sem 14%). At pH = 8.5 (24% of the drug uncharged) the reduction was 73% (sem 30%) . Charge movement remained unaffected by the drug. The pH dependence of the effect together with the indemnity of charge movement suggest that the drug traverses the cellular membrane to reach its binding site, located probably at the SR calcium release channel. Procaine could reduce calcium release by a direct action on the RS calcium release channel or by interfering with a presumed CICR mechanism. Supported with funds of Pedeciba and Fundation Manuel Perez. Differences in hippocampal synaptic plasticity in rats with inborn high or low learning ability may be related to different sensitivity ojaspartate receptors . O. A. RA~z (Depto . de Farmacologia, Fac. de C. Quimicas, UNC.
Côrdoba, Argentina) .
RATS with an inborn high (HP) or low (LP) learning capacity were used to study the sensitivity to the blocking effect of OL-2-amino-5-phosphonovaleric acid (APV-10 and 20 pM) on long-term potentiation (LTP) produced in hippotampal slices by a 1 sec tetanus at 200 Hz . The potential evoked by stimulation of the perforant path was recorded from the granulie cell layer of the dentate gyros in 4001an slices perfused with standard Krebs solution or with APV. Ice all slices from HP rats 10 pM and in 85% of the cases at 20 ~M APV did not block LTP generation . In 60% of slices from LP rats APV 10 kM and in all of the cases at 20 PM APV blocked LTP generation . These results are coherent with the hypothesis that the different inborn learning ability of HP and LP rats is related to the different population or sensitivity of N-methyl-n-aspartate (NMDA) receptors. Syntlresis and M.lO-A inhibitory activity ojsome 4-amine phtnethylmninc derivativts . M. REVes,' R. StLVt~RA,2 F.
DArAS~ and B. CASSets' ('Facultad de Ciencias Universidad de Chile, Santiago, Chile; fileurochemistry Division, IIBCE, Montevideo, Uruguay) . T~ 4amine phenethylamine derivatives (APEAD), 4~imethyhunine amphetamine (4DMAA(`)], [4DMAA(-)]; 4~imethylamine phenethylamine (4DMAPEA) and 4-methylamine amphetamine [4-MMAA(+)], were synthesized and assessed for their monoamine oxidase A inhibitory (MAGI-A) effect. The derivatives were synthesized by condensation of appropriate benzaldehyde with nitromethane or nitroethane in presence of buthyl-amine and further reduction with lithium aluminium hydride. The "in vitro" enzymatic inhibition was tested using a mitochondrial suspension from whole rat brain as source of MAO. 5-HT at appropriate concentrations was utilized as selective substrate for MAO-A. The measurements were performed by HPLC with electrochemical detection using 5-HT and 5-HIAA as markers of enzymatic activity.