George B. Brown

Identification and quantification of 1,2,3,4-tetrahydro-β-carboline, 2-methyl-1,2,3,4-tetrahydro-β-carboline, and 6-methoxy-1,2,3,4-tetrahydro-β-carboline as in vivo constituents of rat brain and adrenal gland☆☆☆

Abstract The identification and quantification of three 1,2,3,4-tetrahydro-β-carbolines as normal constitutents of rat brain and adrenal gland, using combined gas chromatographic/mass spectrometric techniques, are reported. Qualitative analyses of these tissues led to the identification of 1,2,3,4-tetrahydro-β-carboline (THBC), 2-methyl-THBC (2-MTHBC) and 6-methoxy-THBC (6-MeOTHBC), as determined by observed peak retention times, mass fragments and ion mass ratios. Quantitative analyses, using deuterated internal standards, gave the following results: THBC (ng/g wet wt) in brain = 17.5 ± 4.86, adrenal = 500.3 ± 163. 6-MeOTHBC (ng/g wet wt) in brain = 35.6 ± 16.6, adrenal = 1113.7 ± 300. Mechanisms for the formation of these β-carbolines as well as their possible function in vivo are discussed.

Gas chromatographic/mass spectrometric evidence for the identification of 1,2,3,4-tetrahydro-β-carboline as an invivo constituent of rat brain

Abstract Based on gas chromatographic/mass spectrometric data, obtained using the method of selected ion monitoring, the compound 1,2,3,4-tetrahydro-β-carboline has been tentatively identified as an in vivo constituent of rat brain.

Altered brain sodium channel transcript levels in human epilepsy

Normal, and perhaps pathological, characteristics of neuronal excitability are related to the distribution and density of voltage-gated ion channels such as the sodium channel. We studied normal and epileptic human brain using the ligase detection reaction to measure the relative quantities of mRNAs encoding sodium channel subtypes 1 and 2. Normal brains exhibited characteristic 1:2 ratios which varied by brain region, but the ratios were invariate among individuals. These normal values were altered as much as threefold in anatomically corresponding regions of epileptic brain tissues. Changes of this magnitude in such a highly conserved value support a potential role for sodium channels in the pathophysiology of epilepsy.

Batrachotoxinin-A 20-alpha-benzoate: a new radioactive ligand for voltage sensitive sodium channels.

Batrachotoxinin-A 20-α-benzoate (BTX-B), an analog of the potent depolarizing agent batrachotoxin (BTX), was prepared by selective esterification of naturally occurring batrachotoxinin-A with benzoic acid. BTX-B depolarizes rat phrenic nerve-diaphragm preparations with a time course and concentration dependence virtually indistinguishable from that of BTX. A specific, saturable component of equilibrium binding of [3H]BTX-B to mouse cerebral cortex homogenates was measured, described by an equilibrium dissociation constant of 0.7 µM and a maximum number of binding sites of 90 pmol per gram of tissue (wet weight). Specific binding is inhibited by BTX and other BTX analogs, veratridine, and grayanotoxin but is unaffected by tetrodotoxin and cevine. Under conditions of this assay, neither crude Leiurus quinquestriatus scorpion venom nor purified sea anemone toxin have any effect on specific binding. The data support the conclusion that BTX-B interacts with a recognition site associated with voltage sensitive sodium channels which is identical to the recognition site for BTX.

Batrachotoxin: A Window on The Allosteric Nature of The Voltage-Sensitive Sodium Channel

Publisher Summary The voltage-sensitive sodium-channel protein of excitable membranes mediates the fast rising phase of the action potential in a wide variety of tissues. Electrophysiological studies, from the pioneering work of Hodgkin and Huxley onward, have now provided a precise phenomenological description of the sodium channel and the sequence of events occurring at that channel during an action potential. In brief, sodium channels in the resting state of an excitable cell exist in a closed, nonconducting conformation. In response to a local depolarization of the membrane the channel protein undergoes time- and voltage-dependent conformational changes, progressing through apparently multiple nonconducting states before reaching an open, conducting state permitting, selectively, the flow of sodium ions from extracellular to intracellular spaces down an electrochemical gradient. This process is termed “activation.” Thus, binding of batrachotoxin—the prototypical and most potent member of the class—at once affects the voltage-dependent processes of sodium-channel activation and inactivation, changes the selectivity of the channel, and alters the single-channel conductance. This chapter focuses on the body of work that underlies this construct, first from the electrophysiological viewpoint and then from the pharmacological and biochemical approaches. The experiments and the data described here are of necessity selected, rather than all inclusive, with an emphasis placed on batrachotoxin along with veratridine, as these two compounds have been investigated more extensively to date.

Isolation of a human-brain sodium-channel gene encoding two isoforms of the subtype III alpha-subunit.

Voltage-gated sodium channels are members of a multigene family of transmembrane proteins that are important determinants of electrical excitability in cell membranes. These proteins are typically composed of a large α-subunit and one or two β-subunits. The primary structure of α-subunits is highly conserved among different subtypes and different species. Based on the conserved sequences and application of the rapid amplification of cDNA ends (RACE) reaction, we have isolated three overlapping clones from human brain. These sequences share highest homology (89%) to the rat brain subtype III gene and cover a 4.2-kb expanse of the transcript. The 5′-most clone has a translation start site located in the same region as other mammalian brain sodium channel genes. A 92-nucleotide insert was found in domain I at a location previously demarcated by published splice sites in rat brain sodium channels IIN/IIA and IIIN/IIIA. It is most likely that this transcript represents the two isoforms (neonatal and adult) of the human brain sodium channel gene, SCN3A (GenBank accession numbers AF035685 and AF035686). As is the case for rat brain sodium channels IIN/IIA and IIIN/IIIA, these isoforms are generated through an alternative splicing mechanism. The conservation of the exon structure suggests that alternative RNA splicing is a common feature for sodium channel mRNA processing and may play an important role in modulating the channel function.

Differential expression of two sodium channel subtypes in human brain

Two partial human brain sodium channel cDNA sequences (designated HBSC I and II) have been cloned and mapped to chromosome 2q23–2q24 by chromosome microdissection‐PCR (CMPCR). The distribution of HBSC I and II mRNA in human brain was studied by means of a novel approach based on the ligase detection reaction. These studies demonstrate that HBSC I and II mRNA is heterogeneously distributed in brain. and that the relative ratio or the two forms can vary as much as 7‐fold between different regions.

Barbiturate-benzodiazepine interactions at the gamma-aminobutyric acidA receptor in rat cerebral cortical synaptoneurosomes.

Combinations of benzodiazepines (midazolam and diazepam) with barbiturates (pentobarbital and phenobarbital) exhibit synergistic (supra-additive) hypnotic interactions in rats. Because both benzodiazepines and barbiturates interact with the γ-aminobutyric acidA (GABAA) receptor complex, we have tested the hypothesis that these supra-additive hypnotic interactions are due to a synergistic effect on 36CI− conductance subsequent to binding at allosterically coupled sites on the GABAA receptor ionophore complex. Equilibrium binding and 36CI− flux measurements were performed under nearly identical conditions using rat brain cerebrocortical synaptoneuroomes. The benzodiazepines and barbiturates alone both allosterically enhance binding of [3H]muscimol to comparable, but modest, extents (range = 18%–32% enhancement). Isobolographic analysis reveals that combinations of benzodiazepines and barbiturates do in fact produce a synergistic enhancement of [3H]muscimol binding. Paradoxically, this effect is not translated into a synergistic enhancement of muscimolstimulated 36CI− flux. Because the positively cooperative interactions between benzodiazepines and barbiturates, as demonstrated both behaviorally and by binding measurements, are not reflected in enhanced CI− conductance, the mechanistic basis for hypnotic synergism may involve other non-GABAergic components.

γ‐Aminobutyric AcidA Receptor Pharmacology in Rat Cerebral Cortical Synaptoneurosomes

Abstract: Equilibrium binding interactions at the γ‐amino‐butyric acid (GABA) and benzodiazepine recognition sites on the GABAA receptor‐Cl− ionophore complex were studied using a vesicular synaptoneurosome (microsacs) preparation of rat brain in a physiological HEPES buffer similar to that applied successfully in recent GABAergic 36Cr flux measurements. NO 328, a GABA reuptake inhibitor, was included in the binding assays to prevent the uptake of [3H]muscimol. Under these conditions, the equilibrium dissociation constant (Kd) values for [3H]muscimol and [3H]‐diazepam binding are 1.9 μM 40 nM, respectively. Binding affinities for these and other GABA and benzodiazepine agonists and antagonists correlate well with the known physiological doses required to elicit functional activity. This new in vitro binding protocol coupled with 36C1− flux studies should prove to be of value in reassessing the pharmacology of the GABAA receptor complex in a more physiological environment.

Gas chromatographic/mass spectrometric evidence for the identification of 6,7-dihydroxy-1, 2,3,4-tetrahydroisoquinoline as a normal constituent of rat brain: Its quantification and comparison to rat whole brain levels of dopamine

Gas chromatographic/mass spectrometric data are presented which demonstrate the presence of 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (DHTIQ) as a normal constituent of rat brain. The level of DHTIQ was calculated to be 10.0 +/- 3.0 ng/g wet weight (+/- S.D., N = 9) of brain tissue while the level of dopamine (DA) was measured as 1.22 +/- 0.22 microg/g (N = 14). The ratio of DHTIQ:DA was thus observed to be approximately 1:100. The possible formation of DHTIQ in alcoholism and schizophrenia is discussed.