HETEROGENEITY OF HISTAMINE Hi-RECEPTORS: SPECIES VARIATIONS IN [3H]MEPYRAMINE BINDING OF BRAIN MEMBRANES

[³H]Mepyramine binds with high affinity to membranes from brain of human, rat, guinea-pig, rabbit and mouse with drug specificity indicating an association with histamine H₁receptors. Considerable species differences occur in the affinity of [³H]mepyramine, with guinea-pig and human having 34 times greater affinity than rat, mouse or rabbit. The greater affinity of [³H]mepyramine in guinea-pig than in rat is attributable both to faster association and slower dissociation rates in guinea-pig. Species differences in affinity for H₁ receptor sites occur for some antihistamines but not for others. Some tricyclic antidepressant and neuroleptic drugs are extremely potent inhibitors of [³H]mepyramine binding, exceeding in potency any H₁ antihistamines examined. The tricyclic antidepressant doxepin and the neuroleptic clozapine are the most potent of all drugs examined in competing for [³H]mepyramine binding. The regional distribution of specific [³H]mepyramine binding differs considerably in the various species examined.     

STEREOSPECIFIC IN VlTRO BINDING OF [3H]DEXETIMIDE TO BRAIN MUSCARINIC RECEPTORS

A binding assay for muscarinic cholinergic receptors has been developed using labelled dexetimide as ligand and a filtration technique. The main features of this assay are its stereospecific nature, the very high affinity of the ligand for the specific receptors sitcs and its very low affinity for non-specific binding sites. The latter point was further investigated using labelled levitimide, the inactive enantiomer. The binding was found to be neither stereospecific nor saturable and displacement by both enantiomers revealed a particular curve with a very flattened course. Kinetic experiments with [³H]dexetimide suggest the occurrence of a heterogenous population of muscarinic receptors in the rat striatum. A study of the regional distribution of muscarinic receptors in rat brain showed a high concentration in the dopaminergic areas, the cortex and the hippocampus, but practically none in the cerebellum. The subcellular distribution pattern revealed a marked enrichment of [³H]dexetimide stereospecific binding sites in the microsomal fraction of rat striatum and hippocampus. Such a distribution was not found with [³H]levitimide. All the characteristics of this binding assay make dexetimide a very appropriate ligand for labelling muscarinic receptors in vitro as well as in vivo.     

ALTERATIONS IN THE BINDING OF [3H]LEUCINE-ENKEPHALIN TO STRIATAL MEMBRANES BY ENDOGENOUS FACTORS

—Saturation binding studies with [³H]leu-enk ([tyrosyl-3, 5-³H(N)]⁵leu-enkephalin) revealed the presence of high and low affinity binding sites in a paniculate fraction derived from rat striatum. The binding of [³H]leu-enk to the high affinity component (K_D= 2.0 ± 0.3 nM) was sensitive to morphine and levorphanol, while the binding to the low affinity component (K_D= 21 ± 2 nM) was not. Incubation of the membranes, prior to assay for 30 min at 37°C, followed by centrifugation at 27, 000 g for 20 min in order to pellet the membranes allowed the detection of a factor, present in the high speed supernatant, which caused a dose-dependent inhibition of the binding of [³H]leu-enk to the morphine-sensitive and insensitive binding components. Investigations into the nature of the morphine-insensitive binding component demonstrated that it was an artifact since it was not detectable when bound and free ligand were separated by centrifugation. Furthermore, [³H]leu-enk bound to Whatman glass fiber filters, used to collect bound ligand, in a morphine-insensitive manner, and under conditions where the binding of [³H]leu-enk to the morphine sensitive component diluted proportionally with serial dilutions of the membranes, the binding to the morphine-insensitive component did not. The factor present in the high speed supernatant did not dialyze and its effects were mimicked by either trypsin or soybean trypsin inhibitor, but not by bovine serum albumin. The apparent inhibition of the binding of [³H]leu-enk to these binding components is probably not of biological significance, but the fact that the artifactual morphine insensitive binding component of striatal membranes has been shown to decrease by 20–30% following lesions of the substantia nigra suggests that the influence of this endogenous factor must be controlled for.     

THE BINDING OF [3H]MEPYRAMINE TO HISTAMINE H1 RECEPTORS IN GUINEA-PIG BRAIN

The promethazine-sensitive binding of [³H]mepyramine to a membrane fraction from guinea-pig whole brain is saturable with a dissociation constant of 1.7 × 10^-9M. The maximum amount of [³H]mepyramine binding varied widely between preparations, range 122–365 pmol/g protein, with a mean value of 227 ± 52 pmol/g protein. The inhibition of [³H]mepyramine binding by a number of drugs correlated closely with their potency as histamine H₁ antagonists. (+) Chlorpheniramine was 240-fold more potent as an inhibitor of [³H]mepyramine binding than (-)-chlorpheniramine. All antagonists inhibited the binding of [³H]mepyramine to the same extent, but the Hill coefficients characterising the inhibition curves did not all approximate to unity, the value expected for a simple antagonist-receptor equilibrium. The distribution of histamine H₁ receptors, defined by the promethazine-sensitive binding of [³H]mepyramine, in 11 different brain regions was uneven with the largest amounts in cerebellum, superior and inferior colliculus and hypothalamus and the smallest in caudate nucleus, brain stem and spinal cord.     

CHARACTERIZATION OF THE BINDING OF [3H]MUSCIMOL, A POTENT γ-AMINOBUTYRIC ACID AGONIST, TO RAT BRAIN SYNAPTOSOMAL MEMBRANES USING A FILTRATION ASSAY

Abstract— The binding of [³H]muscimol, a potent GABA agonist, to crude synaptic membranes prepared from rat brain was studied using a filtration method to isolate membrane-bound ligand. Specific binding was found to be saturable and occurred to two binding sites of K _d5 5 and 30 n m . Binding was Na⁺-independent and enhanced by both freezing and Triton treatment. Regional and subcellular distribution studies and pharmacological characterization of specific [³H]muscimol binding are consistent with binding to the synaptic GABA receptor.     

HIGH AFFINITY l-[3H]GLUTAMATE BINDING TO POSTSYNAPTIC RECEPTOR SITES ON RAT CEREBELLAR MEMBRANES

l [³H]glutamate binding was investigated in membrane preparations derived from rat cerebellum, an area of the brain where it is likely that a high density of postsynaptic glutamate receptors occurs. Glutamate was hound specifically and, in freshly prepared membranes, was optimal under physiological conditions of pH and temperature and was associated with the synaptic membrane fraction of the cell. Specific binding occurred through a single, high-affinity process with a K_D, of 744 nM and a capacity of 73 pmol/mg protein. Unlike the findings reported for GABA, the specific binding of glutamate to fresh membranes did not involve an uptake site. Comparison of the potencies of a wide range of compounds with known pharmacological activities, demonstrated that their ability to displace specific glutamate binding was consistent with specific interactions with glutamate receptors.     

EFFECTS OF AMINO-OXYACETIC ACID ON [3H]GABA UPTAKE BY RAT BRAIN SLICES

Abstract— The effect of amino-oxyacetic acid on the uptake of [³H]GABA by rat brain slices was studied. When added simultaneously with [³H]GABA, amino-oxyacetic acid had no significant effect on [³H]GABA uptake. However, preincubation of brain slices with amino-oxyacetic acid prior to addition of [³H]GABA produced inhibition of uptake, which increased with longer duration of preincubation. The inhibitory effect of amino-oxyacetic acid was maximal at 2 mM concentration and concentrations sufficient to inhibit significantly GABA:glutamate transaminase (10^--⁶ M) had no effect on [³H]GABA uptake. D-Cycloserine and β-hydrazino-propionic acid also inhibited [³H]GABA uptake, but the amounts required were considerably in excess of those needed to inhibit GABA:glutamate transaminase. 4-Deoxypyridoxine inhibited [³H]GABA uptake, whether given in vivo or in vitro , and the inhibitory effect of amino-oxyacetic acid was reversed with pyridoxine. GABA transport appears to be dependent on pyridoxal phosphate and interference with this function of the vitamin is suggested as the basis for the inhibitory effect of amino-oxyacetic acid on [³H]GABA uptake.     

CHARACTERISTICS AND REGIONAL DISTRIBUTIONS OF TWO DISTINCT [3H]NALOXONE BINDING SITES IN THE RAT BRAIN

Using [³H]naloxone at a concentration of 4.5 nm , the potent opiate agonist etorphine as well as the potent antagonist diprenorphine displace only about 75% of specific naloxone binding P₂ fractions from rat whole forebrain, without additive effect. Several other opiates and antagonists completely displace specific naloxone binding. This indicates that etorphine and diprenorphine specifically bind to one and the same naloxone binding site (type I) while leaving another naloxone binding site (type II) unaffected. Type I binding sites are much more thermo-labile than type II. [³H]Naloxone binding to type I sites is unaffected by incubation temperature in the range 10 to 25°C. while binding type II sites decreases rapidly with increasing incubation temperature, no specific type II binding being detectable at or above 20°C. The two naloxone receptor types also differ with respect to pH dependence, and affinity for naloxone with types I and II having affinity constants (K_d) of 2 and 16 nm , respectively, at 0°C. The two binding sites have different regional distributions with high relative levels of type II receptors in cerebellum and low relative levels in pons-medulla and striatum. In whole rat brain there are about 4 times as many type II receptors as type I. These results suggest that naloxone and several other opiate agonists and antagonists bind to two distinct receptor types which are probably not agonist/antagonist aspects of the same receptor.     

INCREASED BINDING OF [3H]COLCHICINE TO VISUAL CORTEX PROTEINS OF DARK-REARED RATS ON FIRST EXPOSURE TO LIGHT

Abstract— The binding of [³H]colchicine (or a functionally similar metabolite) to acid-insoluble material in vivo was measured in the motor and visual cortices of littermates rats which were either dark-reared (D), exposed to light for 3 h or 24 h (L), or raised normally (N) in 12 h light/12 h dark animal house conditions. No significant differences were found in the binding in the motor cortex of the 3 h or 24 h L, D or N animals, but in the visual cortex after 3 h of light exposure a 23% elevation in binding was measured in L compared with D animals and a small though non-significant (10%) increase in binding was also observed in this region in L compared with N animals. After 24 h of light exposure, binding of the label in the L animals fell near to that of the N and D animals. The results of vinblastine precipitation experiments suggested that much of the radioactivity was bound to the protein tubulin, and this was confirmed when no increased binding of an analogue of colchicine, lumi-colchicine, was observed after 3 h of light exposure in L compared with D animals. It is suggested that these experiments show that colchicine can be used as a marker for changes in the tubulin population in light exposed animals, and demonstrate the transient nature of the increase in tubulin quantity, as opposed to a lasting effect on its synthesis. Further, they argue strongly in support of the idea that a component of protein flow from neuronal cell body to axons and dendrites in light exposed animals, is subject to environmental modification.     

GLYCINE SPECIFIC [3H]STRYCHNINE BINDING IN THE PIGEON CNS

Abstract— Glycine sensitive [³H]strychnine binding has been demonstrated in the pigeon CNS. The kinetic parameters in spinal cord, tectum, telencephalon and cerebellum in this order were: K _n, (nM) 5 , 5, 2.3, 6 and B _max (fmol/mg protein) 1000, 275, 92, 20. The effect of various amino acids and strychnine analogs on the binding was measured. Retinal ablation caused a 22% decrease in the glycine sensitive [³H]strychnine binding in the tectum while lesions destroying up to 80% of the nucleus Ipc had no effect.     

Heterogeneity of [3H]Dipyridamole Binding to CNS Membranes: Correlation with [3H]Nitrobenzylthioinosine Binding and [3H]Uridine Influx Studies

The relationship between the nucleoside transport system and the nitrobenzylthioinosine-sensitive and -resistant [3H]dipyridamole binding sites was examined by comparing the characteristics of [3H]dipyridamole binding with those of [3H]nitrobenzylthioinosine binding and [3H]-uridine influx in rabbit and guinea pig cerebral cortical synaptosomes. Two distinct high-affinity synaptosomal membrane-associated [3H]dipyridamole binding sites, with different sensitivities to inhibition by nitrobenzylthioinosine, were characterized in the presence of 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS, 0.01%) to prevent [3H]dipyridamole binding to glass tubes and filters. The nitrobenzylthioinosine-resistant [3H]-dipyridamole binding sites represented a greater proportion of the total membrane sites in guinea pig than in rabbit (40 vs. 10% based on inhibition studies). In rabbit, nitrobenzylthioinosine-sensitive [3H]dipyridamole binding (KD = 1.4 +/- 0.2 nM) and [3H]nitrobenzylthioinosine binding (KD = 0.30 +/- 0.01 nM) appeared to involve the same membrane site associated with the nitrobenzylthioinosine-sensitive nucleoside transporter. By mass law analysis, [3H]-dipyridamole binding in guinea pig could be resolved into two components based on sensitivity to inhibition by 1 microM nitrobenzylthioinosine. The nitrobenzylthioinosine-resistant [3H]dipyridamole binding sites were relatively insensitive to inhibition by all of the nucleoside transport substrates and inhibitors tested, with the exception of dipyridamole itself. In guinea pig synaptosomes, 100 microM dilazep blocked nitrobenzylthioinosine-resistant [3H]uridine transport completely but inhibited the nitrobenzylthioinosine-resistant [3H]dipyridamole binding component by only 20%. Furthermore, a greater percentage of the [3H]dipyridamole binding was nitrobenzylthioinosine resistant in guinea pig compared with rabbit, yet both species had a similar percentage of nitrobenzylthioinosine-resistant [3H]uridine transport.(ABSTRACT TRUNCATED AT 250 WORDS)     

POTASSIUM-INDUCED RELEASE OF [3H]GABA AND OF [3H]NORADRENALINE FROM NORMAL AND RESERPINIZED RAT BRAIN CORTEX SLICES. DIFFERENCES IN CALCIUMDEPENDENCY, AND IN SENSITIVITY TO POTASSIUM IONS

The release of [³H]GABA induced by elevated extracellular potassium (K)_o, from thin rat brain cortex slices, has been compared with that of [³H]noradrenaline ([³H]NA), released by the same procedures, both from normal slices, and from slices pre-treated with reserpine and nialamide, [³H]NA being predominantly a vesicular component in the former situation, and a soluble substance in the latter one. 46 mM-(K)_o released considerably more [³H]NA from normal than from drug-treated slices, while the release of GABA was about two thirds of the latter. When 4min ‘pulses’ of increasing concentrations of potassium were applied, it was observed that the release of GABA and of [³H]NA from drug-treated slices increased in proportion to (K)_o, up to 36-46 mM and then declined considerably with higher (K)_o. The dependency of potassium-induced release on the concentration of calcium in the medium, indicated that release of [³H]NA from normal slices was proportional to calcium up to 1.5-2 mM, while that of [³H]NA from drug-treated slices increased up to 0.5 mM-Calcium, and then declined with higher concentrations. GABA release also increased up to 0.5 mM-calcium, but no further changes were observed at higher concentrations. The calcium antagonist D-600 inhibited high (K)_o-induced release of [³H]NA from normal slices to a greater extent than that of [³H]GABA or of [³H]NA from drug-treated slices. These results, in which elevated (K)_o-induced release of [³H]GABA resembles considerably that of soluble NA, but differs from that of NA present in synaptic vesicles, suggest that release of [³H]GABA also occurs from the soluble cytoplasmic compartment, and that the partial calcium requirement that is found is unrelated to that of transmitter secretion. These findings are also a further indication of the lack of specificity of elevated (K)_o as a stimulus for inducing transmitter secretions.     

Binding Sites for [3H]Glutamate and [3H]Aspartate in Human Cerebellum

Abstract The binding of [³H]aspartate and [³H]glutamate to membranes prepared from frozen human cerebellar cortex was studied. The binding sites differed in their relative proportions, their inhibition by amino acids and analogues, and by the effects of cations. A proportion (about 30%) of [³H]glutamate binding was to sites similar to those labelled by [³H]aspartate. An additional component of [³H]gluta-mate binding (about 50%) was displaced by quisqualate and aL-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, and may represent a “quisqualate-preferring” receptor. Neither N-methyl-d-aspartic acid-sensitive nor dl-2-amino-4-phosphonobutyric acid-sensitive [³H]glutamate binding was detected.     

ISOLATION OF [3H]ACETYLCHOLINE POOLS BY SUBCELLULAR FRACTIONATION OF CEREBRAL CORTEX SLICES INCUBATED WITH [3H]CHOLINE

Abstract— Subcellular fractions were isolated from tissue incubated in [³H]choline with or without the addition of 33 mM-KCl. Radioactive and bioassayable ACh were measured in the synaptosomes, synaptosomal cytoplasm and in the vesicles. After incubation with KCI the vesicles, as isolated, contained ACh of a lower specific activity than the cytoplasmic ACh. Therefore the vesicle fraction as isolated does not represent the source of the high specific activity ACh released upon K⁺ stimulation. However the vesicle fraction is heterogeneous. Most of the bioassayable ACh but little of the radioactive ACh in the vesicles passed through iso-osmotic Sephadex columns. These results raise the question of the existence of vesicles which contain highly radioactive ACh but which lose it during their isolation by current methods. Different possible forms of heterogeneity are discussed.     

CHEMICAL SYNTHESIS OF [3H]CEREBROSIDE [35S]SULFATE AND ITS IN VIVO METABOLISM IN RAT BRAIN

—Double-labeled sulfatide containing [3-³H]lignoceric acid and [³⁵S]sulfate was synthesized and injected intracerebrally into 28-day-old rats. The ³H-labeled sulfatide was synthesized by condensing (RS)-[3-³H]lignoceroyl chloride with lysosulfatide which had been obtained by saponification of sulfatide. The ³⁵S-labeled sulfatide was synthesized by using [³⁵S]sulfuric acid for sulfating 2′, 4′, 6′-tri-benzoyl-galactosyl N-fatty acyl, N-benzoyl-3-0-benzoyl-sphingosine, which had been obtained by per-benzoylation followed by solvolysis of calf brain nonhydroxycerebrosides. The perbenzoylated [³⁵S]sul-fatide was then subjected to mild alkaline saponification. Eight hours following the injection, the brain lipids contained various radioactive sphingolipids in addition to sulfatides. Fourteen per cent of the injected ³H was recovered in total lipids, and 26% of this was found in sulfatide. Nonhydroxy- and hydroxyceramides, nonhydroxy- and hydroxycerebrosides, and polar lipids contained 7, 1, 8, 3, and 22 per cent of the ³H found in total lipids, respectively. On the other hand, only 6% of the ³⁵S injected was recovered in total lipids; 63% of this was found in sulfatide, 5% in a mixture of seminolipid and cholesterol sulfate and 10% in a water-soluble material.     

THE EFFECT OF DELTA-AMINOLAEVULINIC ACID ON THE UPTAKE AND EFFLUX OF [3H]GABA IN RAT BRAIN SYNAPTOSOMES

§-Aminolaevulinic acid (§-ALA) is an omega amino acid which can be considered as an analogue of γ-aminobutyric acid (GABA). We have examined the effect of §-ALA on [³H]GABA uptake and release in the synaptosome fraction of rat cerebral cortex and report: (1) High concentrations of §-ALA (0.75-5 mM) stimulated [³H]GABA release very markedly, the stimulation with 1mM and 5mM-§-ALA exceeding the maximum obtainable with unlabelled GABA; (2) Low concentrations of §-ALA (0.1-0.5 mM) produced little stimulation of [³H]GABA efflux, less than that produced by similar concentrations of unlabelled GABA; (3) 0.1 mM-§-ALA reduced the stimulation of [³H]GABA efflux elicited by 55 mM-K⁺ and the combination of 1 mM-§-ALA and 55mM-K⁺ produced a lower stimulation of efflux than 1 mM-§-ALA alone; (4) §-ALA inhibits [³H]GABA uptake in a linearly competitive fashion and inhibition is maximal at 0.5 mM-§-ALA. These results are discussed in relation to the neuronal high affinity GABA transport mechanism and inhibition of the synaptosomal Na⁺ and K⁺ -dependent ATPase. It is also postulated that §-ALA increases the chloride conductance of the synaptosomal membrane, possibly by acting on presynaptic GABA receptors.     

ON THE RELATIONSHIP BETWEEN [3H]CHOLINE UPTAKE ACTIVATION AND [3H]ACETYLCHOLINE RELEASE

The depolarization-induced, calcium-dependent release of [³H]ACh from hippocampal synaptosomes was studied in a superfusion system. Release increased, with increasing depolarization. Barium and strontium effectively substituted for calcium during the depolarization, but magnesium inhibited the release. Releasable [³H]ACh is derived from the sodium-dependent component of the [³H]choline uptake which points out the physiologic importance of sodium-dependent choline transport. It is concluded that [³H]ACh release in this system has the same properties as neurotransmitter release in many other systems. Previous studies have shown that treatments which alter the activity of cholinergic neurons in vivo result in parallel changes in sodium-dependent choline uptake in vitro. When synaptosomes were utilized from animals treated to reduce cholinergic activity, there was a reduced release following the reduced uptake. Conversely, when synaptosomes were taken from animals treated to increase sodium-dependent choline uptake, there was an increase in the release. It is concluded that the changes in sodium-dependent choline uptake in vitro consequent to changes in neuronal activity in vivo result in parallel changes in releasable ACh. A comparison was made between the effect of a number of ions and agents on release and their effect on the in vitro, depolarization-induced activation of sodium-dependent choline uptake. Barium and strontium, ions which substitute for calcium in the release process, support the in vitro activation of uptake. Vinblastine and Bay a 1040, compounds which block release, prevented the in vitro activation of sodium-dependent choline uptake. However, magnesium blocked release in a dose-dependent manner, but did not block the activation of uptake in vitro. Rather, magnesium substituted for calcium and supported the activation of uptake in a dose-dependent fashion. It is concluded that acetylcholine release is not necessary for the activation of choline uptake.     

[3H]GLYCOGEN HYDROLYSIS IN BRAIN SLICES: RESPONSES TO NEUROTRANSMITTERS AND MODULATION OF NORADRENALINE RECEPTORS

Abstract— Different agents have been investigated for their effects on [C³H]glycogen synthesized in mouse cortical slices. Of these noradrenaline, serotonin and histamine induced clear concentration-dependent glycogenolysis.
[C³H]Glycogen hydrolysis induced by noradrenaline appears to be mediated by beta-adrenergic receptors because it is completely prevented by timolol, while phentolamine is ineffective. It seems to involve cyclic AMP because it is potentiated in the presence of isobutylmethylxanthine; in addition dibutyryl cyclic AMP (but not dibutyryl cyclic GMP) promotes glycogenolysis.
Lower concentrations of noradrenaline were necessary for [C³H]glycogen hydrolysis (EC₅₀= 0.5μM) than for stimulation of cyclic AMP accumulation (EC₅₀= 8μM).
After subchronic reserpine treatment the concentration-response curve to noradrenaline was significantly shifted to the left (EC₅₀= 0.09 ± 0.02 μM as compared with 0.49 ± 0.08 μM in saline-pretreated mice) without modifications of either the basal [C³H]glycogen level, maximal glycogenolytic effect, or the dibutyryl cAMP-induced glycogenolytic response.
In addition to noradrenaline, clear concentration-dependent [³H]glycogen hydrolysis was observed in the presence of histamine or serotonin. In contrast to the partial [³H]glycogen hydrolysis elicited by these biogenic amines, depolarization of the slices by 50 mM K⁺ provoked a nearly total [C³H)glycogen hydrolysis.     

THE EFFECT OF PHOSPHOLIPASE C, PHOSPHOLIPASE A2 AND NEURAMINIDASE ON THE UPTAKE OF [3H]NOREPINEPHRINE AND [3H]SEROTONIN

Abstract— The uptake of [³H]norepinephrine ([³H]NE) and [³H]serotonin ([³H]5-HT) by rat brain synaptosomes is reduced as a result of pretreatment of the synaptosomes with phospholipase C (EC 3.1.4.3) or phospholipase A₂ (EC 3.1.1.4). This effect is not due to inhibition of the Na⁺-K⁺-ATPase but rather is caused by hydrolysis of neuronal membrane phospholipids, mainly phosphatidylcholine, which seem to be important to the uptake.