Differential binding of 3H-imipramine and 3H-mianserin in rat cerebral cortex

Drug competition profiles, effect of raphé lesion, and sodium dependency of the binding of two antidepressant drugs ³H-imipramine and ³H-mianserin to rat cerebral cortex homogenate were compared to examine whether the drugs bound to a common “antidepressant receptor.” Of the neurotransmitters tested, only serotonin displaced binding of both ³H-imipramine and ³H-mianserin. ³H-mianserin binding was potently displaced by serotonin S₂ antagonists and exhibited a profile similar to that of ³H-spiperone binding. In the presence of the serotonin S₂ antagonist spiperone, antihistamines (H₁) potently displaced ³H-mianserin binding. ³H-Imipramine binding was displaced potently by serotonin uptake inhibitors. The order of potency of serotonergic drugs in displacing ³H-imipramine binding was not similar to their order in displacing ³H-spiperone or ³H-serotonin binding. Prior midbrain raphé lesions greatly decreased the binding of ³H-imipramine but did not alter binding of ³H-mianserin. Binding of ³H-imipramine but not ³H-mianserin was sodium dependent. These results show that ³H-imipramine and ³H-mianserin bind to different receptors. ³H-Imipramine binds to a presynaptic serotonin receptor which is probably related to a serotonin uptake recognition site, the binding of which is sodium dependent. ³H-Mianserin binds to postsynaptic receptors, possibly both serotonin S₂ and histamine H₁ receptors, the binding of which is sodium independent.     

Stimulatory effect of somatostatin on norepinephrine release from rat brain cortex slices

The effect of somatostatin (SRIF) on norepinephrine (NE) release from the brain tissue was determined on the superfused rat cerebral cortex slices preloaded with ³HNE. SRIF (0.38 μM–1.53 μM) was found to stimulate dose-dependently tritium (³H) overflow evoked electrically by 30%—116% although SRIF did not affect on the spontaneous ³H overflow. SRIF at the concentrations which exhibited the stimulatory effect inhibited scarecely the uptake of ³HNE by cortex slices, while the reference drug, cocaine (50 μM, 10 μM) markedly depressed the uptake. The stimulatory effect of SRIF was not reduced by phentolamine (3.14 μM), α-adrenoceptor blocker, which increased the evoked ³H overflow from the slices itself. These results suggest that SRIF does not produce its stimulatory effect by inhibiting the NE reuptake mechanisms or by interacting with the presynaptic α-adrenoceptors. Elevating of Ca²⁺ concentrations from 0.75 mM to 2.25 mM in the superfusion fluid reduced the stimulatory effect of SRIF. It is possible that SRIF stimulates NE release by facilitating the availability of Ca²⁺ for the release mechanisms.     

3H-clozapine binding to rat brain membranes

³H-Clozapine binds specifically and with high affinity (K_D = 1.3 nM) to rat brain membranes. About two thirds of reversibly bound ³H-clozapine are displaced by hyoscyamine in a stereospecific manner, suggesting interaction of clozapine with muscarinic cholinergic receptors. Most of the remaining ³H-clozapine binding is stereospecifically inhibited by butaclamol, but this binding component seems not to be related to dopamine receptors.     

Depolarization-evoked release of gamma-hydroxybutyrate from rat brain slices

The release of gamma-hydroxybutyrate from preloaded rat brain striatal slices was investigated. K+-induced depolarization caused an efflux of gamma-hydroxybutyrate of about 50 fmol min-1 mg-1 (wet weight), but in a Ca2+-free medium containing Mg2+, the evoked release was reduced by 50-60%. The release was higher when 100 microM veratridine was used as a depolarizing agent. The efflux of gamma-hydroxybutyrate is related to veratridine and K+ concentration, and is strongly inhibited by 10 microM tetrodotoxin. The Ca2+ channel blocker verapamil induces a large decrease in the efflux of gamma-hydroxybutyrate after both K+- and veratridine-induced depolarization. These results are in favour of a possible transmitter function for gamma-hydroxybutyrate in rat striatum.     

Depolarization-induced release of [3H]histamine by high potassium concentrations,electrical stimulation and veratrine from rat brain slices after incubation with the radiolabelled amine

Brain slices obtained from neocortex, hypothalamus or hippocampus were incubated with [³H]histamine and subsequently superfused and exposed to different depolarizing stimuli, viz. high K⁺-concentrations, electrical field stimulation and veratrine. K⁺-induced release of tritium was completely calcium-dependent and its magnitude depended on the K⁺-concentration, with maximal release being reached at 56 mM K⁺. Electrically-evoked release of tritium increased with increasing frequencies and reached its maximum at about 20 Hz. The electrically-evoked release appeared to be totally calcium-dependent and it was strongly inhibited by tetrodotoxin. Veratrine (5–100 μM) also induced a release of tritium; maximal release was obtained at 100 μM veratrine. Veratrine-induced release was partially calcium-dependent and was strongly reduced by tetrodotoxin.Taken together the data indicate that the depolarization-induced release of tritium from brain slices pre-labelled with [³H]histamine, represents [³H]histamine release from neurons and not from either mast cells or glial cells. It remains to be established whether these neurons are specifically histaminergic.     

Tryptamine transport in rat brain slices: A comparison with 5-hydroxytryptamine

The uptake of [¹⁴C]tryptamine (¹⁴C-T) and [³H]serotonin (³H-5HT) into slices of rat hypothalamus (HT), fronto-parietal cortex (CX), and caudate nucleus (Cau) has been investigated. In all three brain areas, the uptake of³H-5HT at 37°C was much greater than that in an ice-bath at 1.0–1.5°C. In contrast, the uptake of¹⁴C-T at 37°C was not much greater than uptake at 1.0–1.5°C. While markedly different amounts of³H-5HT were accumulated by each of the brain areas studied, the regional uptake of¹⁴C-T was quantitatively similar. In general the uptake of¹⁴C-T was inhibited less than³H-5HT by cocaine, DNP, ouabain, and decreased Na⁺ concentrations. Similarly,¹⁴C-T was less susceptible to serotonin uptake inhibitors except in the caudate. It was concluded that though a common indoleamine uptake system accumulates both T and 5HT, a non-specific low affinity or diffusional process also transports both amines and is predominantly responsible for T, but not 5HT, uptake. The spontaneous release, or wash-out, of¹⁴C-T from the caudate was much faster than that of³H-5HT. In addition, while depolarizing stimuli caused little or no release of¹⁴C-T, large releases of³H-5HT were observed. T, therefore, does not behave like a conventional neurotransmitter.     

Serotonin-1B receptor-mediated inhibition of [3H]GABA release from rat ventral tegmental area slices

In order to assess a role of 5-HT(1B) receptors for regulation of GABA transmission in the ventral tegmental area (VTA), VTA slices from the rat were incubated with [(3)H]GABA and beta-alanine, and superfused in the presence of nipecotic acid and aminooxyacetic acid. [(3)H]GABA release was induced by exposures to the medium containing 30 mM potassium for 2 min. The results showed that high potassium-evoked [(3)H]GABA release was sensitive to calcium withdrawal or blockade of sodium channels by tetrodotoxin, suggesting that tritium overflow induced by high potassium derived largely from neuronal stores. Administration of CP 93129 (0.15 and 0.45 microM), a 5-HT(1B) receptor agonist, or RU 24969 (0.15 and 0.45 microM), a 5-HT(1B/1A) receptor agonist, but not 8-OH-DPAT (0.45 microM), a 5-HT(1A) receptor agonist, inhibited high potassium-evoked [(3)H]GABA release in a concentration-related manner. The RU 24969-induced inhibition of [(3)H]GABA release was antagonized by either SB 216641, a 5-H(1B) receptor antagonist, or cyanopindolol, a 5-HT(1B/1A) receptor antagonist, but not by WAY 100635, a 5-HT(1A) receptor antagonist. Pre-treatment with SB 216641 also antagonized CP 93129-induced inhibition of [(3)H]GABA release. The results support the hypothesis that 5-HT(1B) receptors within the VTA can function as heteroreceptors to inhibit GABA release.     

3H-dopamine uptake by synaptic storage vesicles of rat whole brain and brain regions

A crude preparation of neurotransmitter storage vesicles was obtained by differential centrifugation and the ability to take up ³H-dopamine was evaluated $\text{in}$$\text{vitro}$. The uptake was highly dependent on temperature, had an absolute requirement for ATP and Mg²⁺ and was inhibited totally by reserpine. The uptake displayed saturation kinetics, with a Km of 0.26 μM at 20°. ³H-dopamine uptake was inhibited competitively by norepinephrine, with a Ki of 0.69 μM. Vesicles derived from a primarily dopaminergic region (corpus striatum) exhibited the same ratio of uptakes of ³H-dopamine/³H-norepinephrine as did those from a primarily noradrenergic region (cerebral cortex). These results indicate that viable rat brain storage vesicles can be readily prepared and used for evaluation of pharmacologic effects on ³H-dopamine uptake, and that dopaminergic and noradrenergic storage vesicles exhibit identical uptake properties.     

Properties of 3H-cimetidine binding in rat brain membrane fractions

In an attempt to characterize the brain histamine H₂ receptor, experiments were undertaken to study the binding properties of (N-methyl-³H) -cimetidine, an H₂ receptor antagonist, in rat brain membranes. Using a centrifugation assay, ³H-cimetidine binding having a K_d of 0.40μM and a Bmax of 3.9 pmoles/mg protein was detected. Of fourteen anions and cations tested, one, Cu⁺⁺, dramatically increased specific ³H-cimetidine binding, the increase being due mainly to a change in Bmax. Studies of substrate specificity for ³H-cimetidine binding revealed that Cu⁺⁺, while not significantly affecting the potency of H₂ receptor agonists and antagonists, dramatically decreases the potency of H₁ receptor substances on the ³H-cimetidine binding site. In addition, both the relative and absolute potencies of various H₂ receptor agonistsv and antagonists in displacing the ligand in the presence of Cu⁺⁺ parallels their potencies in biological systems. These findings suggest that, under these conditions, ³H-cimetidine may be labelling a biologically relevant H₂ binding site in brain and that Cu⁺⁺ may regulate the substrate specificity for this site. The brain regional distribution and kinetic analysis of the binding suggest that it is not localized solely to the synaptic receptor for histamine, but may also be associated with histamine receptors at other neuronal, glial or vascular sites.     

2H- and 31P-NMR studies of bilayers composed of 1-acyllysophosphatidylcholine and fatty acids

1-Palmitoyllysophosphatidylcholine has been mixed in equimolar amounts with specifically deuterated palmitic acid and the structural properties of the lipid/water phase have been studied by ²H- and ³¹P-nuclear magnetic resonance. The order profile of the free palmitic acid is very similar to that of the parent compound $\text{1,2-}\text{dipalmitoyl}\text{-sn-}\text{glycero-3-phosphocholine}$ at temperatures above the gel-to-liquid crystal phase transition. The bending of the $\text{sn-2}$ chain which is typical for diacyl lipids is not observed for the free palmitic acid. The mixture of lysolipid and palmitic acid exhibits well-defined quadrupole splittings even at temperatures below the gel-to-liquid crystal phase transition. Hence it is possible for the first time to establish an order profile in the gel-state of the lipid bilayer phase. Between carbon atoms 5 to 12 the palmitic acid chain is found to assume the extended all-trans conformation with a very small contribution from gauche defects. Towards the methyl terminal a distinct increase in the gauche probability can be noted. The motion of the phosphocholine headgroup was also studied by ²H- and ³¹P-NMR using selectively deuterated 1-palmitoyllysophosphatidylcholine. The headgroup has a considerably larger motional freedom in the mixture of lysolipid and palmitic acid than in $\text{1,2-}\text{dipalmitoyl}\text{-sn-}\text{glycero-3-phosphocholine}$. In addition, the average headgroup conformations are also different in the two systems.     

Evoked release of tissue-bound exogenous [1-14C]acetylcholine from rat brain cortex slices

Exogenous labeled acetylcholine ([¹⁴C]ACh) bound, in rat brain cortex slices, in a poorly (or non-) exchangeable form, by prior incubation of the slices in presence of 5 mM [¹⁴C]ACh, is partly released in an ACh-free physiological saline-glucose-paraoxon medium by a variety of conditions. Among these are high [K⁺], lack of Na⁺ or Ca²⁺, and the presence of protoveratrine or ouabain. The releasing effect of protoveratrine is completely abolished by tetrodotoxin which itself is without effect. Only about half of the retained or tissue-bound [¹⁴C]ACh is affected by these conditions. The whole of the bound ACh is released by treatment with acid or by dissolution of the cell membranes. The stimuli that release part of the bound exogenous [¹⁴C]ACh appear to be similar to those that release glucose-derived tissue-bound ACh formed during normal cerebral metabolism.     

Determination of the complete order parameter tensor for a lipid methylene group from 1H- and 2H-NMR spin labels

Proton-proton dipolar splittings are obtained as a function of temperature for the α-methylene in a potassium palmitate - (β-ω) - d₂₉ (70 wt.%) / D₂O (30 wt.%) sample above and below the gel to liquid crystal phase transition. These splittings and corresponding deuteron quadrupole splittings are used to specify the complete order parameter tensor for the α-methylene group. Deduction of lipid structural information from the complete-order parameter tensor is discussed.     

Identification of 5-hydroxytryptamine binding substances from rat brain stem

Abstract— Godwin & Sneddon (1975) reported the binding of 5-hydroxy-[³H]tryptamine (5-HT) on a Sephadex LH-20 column to‘proteolipid material’extracted with n-butanol from rat brain stem. An examination of this‘proteolipid material’with TLC showed the main constituents to be cerebroside sulfate (CS), monophosphoinositide (PI), and diphosphoinositide. The elution profiles of [³H]5-HT incubated with purified CS or with a mixture of CS and PI were similar to that of the brain extract on the same column. Because the elution profile of the mixture of CS and PI was more similar to that of the brain extract, it was concluded that what was suggested to be a possible proteolipid‘5-HT receptor’was mainly two acidic lipids. The elution profile of [³H]5-HT incubated with purified PI, however, was similar to [³H]5-HT eluted alone. This suggested that either PI did not bind to 5-HT or that the PI-5-HT complex possesses different Chromatographie behavior than PI. To test this latter possibility, [¹⁴C]5-HT and [³H]PI were incubated then eluted on a Sephadex LH-20 column with a continuous gradient of increasing polarity. The gradient first eluted PI, then an apparent PI-5-HT complex, and finally 5-HT. This demonstrated that PI will bind to 5-HT on a Sephadex LH-20 column and that the PI-5-HT complex is probably more polar than PI.     

Depolarizing and calcium-mobilizing stimuli fail to enhance synthesis and release of endocannabinoids from rat brain cerebral cortex slices

The concentrations of the endocannabinoids 2-arachidonoylglycerol (2-AG) and N-arachidonylethanolamine (anandamide) were examined in rat brain cerebral cortex slices and surrounding medium. Basal concentrations of endocannabinoids were similar to those identified previously in rat brain, with anandamide content being much lower (19 pmol/g) than that of 2-AG (7300 pmol/g). In contrast, basal concentrations in the surrounding medium were proportionally much lower for 2-arachidonoylglycerol (16 pmol/mL) compared to anandamide (0.6 pmol/mL). Incubation of slices with glutamate receptor agonists, depolarizing concentrations of KCl, or ionomycin failed to alter tissue concentrations of endocannabinoids, while endocannabinoids in the medium were unaltered by elevated KCl. Cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester, an inhibitor of fatty acid amide hydrolase, significantly enhanced tissue concentrations of anandamide (and related N-acylethanolamines), without altering 2-AG, while evoking proportional elevations of anandamide in the medium. Removal of extracellular calcium ions failed to alter tissue concentrations of anandamide, but significantly reduced 2-AG in the tissue by 90% and levels in the medium to below the detection limit. Supplementation of the medium with 50 μM N-oleoylethanolamine only raised tissue concentrations of N-oleoylethanolamine in the presence of cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester and failed to alter either tissue or medium anandamide or 2-AG concentrations. These results highlight the ongoing turnover of endocannabinoids, and the importance of calcium ions in maintaining 2-AG concentrations in this tissue.     

Effect of barbiturates on release of endogenous amino acids from rat cortex slices

The potassium-stimulated, calcium-dependent release of endogenous GABA and glutamic acid was suppressed by pentobarbital. The ouabain and veratridinestimulated fluxes of the amino acids, calcium-independent processes, were not suppressed by pentobarbital. Release of GABA and glutamic acid was not suppressed by pentobarbital in the presence of the calcium ionophore A23187. Of eight barbiturates studied at equimolar concentrations six were found to inhibit GABA release. Thiopental was the most potent, and phenobarbital and secobarbital were inactive.