Quantitative assessment of heterogeneous 3H-spiperone binding to rat neostriatum and frontal cortex

Anomalies of the binding of ³Hspiperone to rat cerebral membranes have been examined. By employing a very low ligand concentration ($\text{～ 25}\text{pM}{}^3\text{Hspiperone}$) we have demonstrated that even within the corpus striatum, ³Hspiperone appears to bind to multiple sites and that dopaminergic and serotonergic agents can selectively inhibit from these sites. In the corpus striatum, 75–80% of the ³Hspiperone specific binding can be inhibited with high affinity by dopaminergic drugs while some 20–30% is inhibited with high affinity by serotonergic compounds. The two ³Hspiperone sites, which we have shown to have affinities of 31 and 325 pM, may therefore represent dopaminergic and serotonergic sites. At higher concentrations of ³Hspiperone, however, the picture may be complicated by a further low affinity site. The great selectivity shown by dopaminergic agonists for the two ³Hspiperone sites explains the ‘flattened’ displacement curves reported for ³Hspiperone/agonist interactions. As dopaminergic agents show the greater affinity for the high affinity ³Hspiperone site, it is tempting to speculate that this site has the greatest association with the dopamine receptor.     

The isomers of cocaine and tropacocaine: Effect on 3H-catecholamine uptake by rat brain synaptosomes

Compared to (+)-$\text{pseudo}$cocaine, (?)-cocaine was 20 times more potent in inhibiting uptake of ³H-norepinephrine (³HNE) by cortical synaptosomes and 66 times more potent with respect to ³H-dopamine (³HDA) uptake by striatal synaptosomes. Although the tropacocaine isomers were equipotent as inhibitors of ³HNE uptake in the cortex, tropacocaine was 3.9 times more potent as an inhibitor of ³HDa uptake in the striatum than $\text{pseudo}$tropococaine. A major known cocaine metabolite, benzoylecgonine failed to inhibit the accumulation of ³HNE and ³HDA by synaptosomes from the cortex and striatum, respectively. The implications of these findings in relation to the motor stimulation seen with (?)-cocaine, (+)-$\text{pseudo}$cocaine and benzoylecgonine in rats are discussed.     

Chronic ethanol alters the receptor binding characteristics of the delta opioid receptor ligand,DAla2DLeu5 enkephalin in mouse brain

The binding characteristics of the delta opioid receptor ligand, ³HDAla²DLeu⁵ enkephalin, were markedly altered in brains obtained from mice fed an ethanol-containing diet for five days. Control mice exhibited both a high and low affinity site for ³HDAla²DLeu⁵ enkephalin, whereas those consuming the ethanol diet were found to possess only one binding site. This singular site has an intermediate K_D value with an increase in receptor number when compared to the high and low affinity sites observed in control mice. The $\text{in}$$\text{vitro}$ addition of ethanol to a brain membrane preparation obtained from untreated mice, at a concentration equivalent to that found in the blood of the ethanol-treated mice, did not markedly affect DAla²DLeu⁵ enkephalin binding characteristics. No alteration in the binding characteristics of ³H-naloxone, a mu receptor ligand, was noted following five days of ethanol consumption. Mice maintained on the ethanol-containing diet were tolerant to the activity-stimulating effects of acute ethanol administration. These results suggest that mice consuming an ethanol diet in sufficient quantities to render them tolerant exhibit a specific loss of a ³HDAla²DLeu⁵ enkephalin binding site, while the binding of ³H-naloxone was unchanged.     

The influence of temperature and gamma-aminobutyric acid on benzodiazepine receptor subtypes in the hippocampus of the rat

The influence of GABA on the affinity of flunitrazepam (FLU) for benzodiazepine receptor subtypes (type I and II) was studied by measurement of the competitive inhibition of [³H]FLU and [³H]propyl beta-carboline-3-carboxylate ([³H]PCC) binding. When assays were carried out at 0°C using a low concentration (0.040 nM) of [³H]PCC so that the type I receptors were selectively labelled, no significant effect of GABA (10^?4 M) on the $\text{FLU}\text{[}{}^3\text{H]}\text{PCC}$ competition curve was detected. In contrast, when assays were carried out at 0°C using [³H]FLU or a high concentration of [³H]PCC to achieve [³H]ligand receptor occupancy of both type I and type II receptors, GABA (10^?4 M) caused a significant increase in the affinity of FLU as measured by $\text{FLU}\text{[}{}^3\text{H]}\text{FLU}$ and $\text{FLU}\text{[}{}^3\text{H]}\text{PCC}$ competition experiments. Collectively, these data suggest that the influence of GABA on benzodiazepine receptor binding is mediated, primarily, by the type II receptor. It was also noted that the $\text{PCC}\text{[}{}^3\text{H]}\text{FLU}$ competition curve had a Hill coefficient of approximately 1 at 37°C as compared to the results of experiments at 0°C during which a Hill coefficient of approximately 0.7 was calculated.     

Antinociceptive and hypothermic effects of trimethyltin

Trimethyltin (TMT) induced a dose-dependent antinociceptive and hypothermic effect in mice. Antinociception was not attenuated by naloxone but was reversed by atropine. TMT, however, was ineffective in displacing (³H)-QNB binding $\text{in vitro}$ and did not affect (³H)-QNB binding or acetylcholinesterase activity after $\text{in vivo}$ administration. The ethyl ester of nipecotic acid, a specific inhibitor of synaptosomal GABA uptake, exerted a similar antinociceptive effect that could be blocked by atropine. The GABA receptor antagonist bicuculline attenuated antinociception induced by TMT and nipecotic acid ethyl ester but not by morphine or oxotremorine. γ-Vinyl GABA, an irreversible inhibitor of GABA metabolism, prolonged TMT but not morphine-induced antinociception. In contrast, neither the dose-response nor the time course of TMT-induced hypothermia were affected by any of the drugs tested. The findings suggest that the GABAergic system may be involved in TMT induced antinociception; however, the mechanism responsible for the hypothermic effect of TMT is not apparent.     

Laser-Raman study of valinomycin-doped and omega-CD3-labeled phosphatidylcholine liposomes

The relative intensities of the CH stretching vibrations are used to study the interaction of lecithin liposomes with valinomycin, a mobile carrier for alkali ions. In the case of dipalmitoyl lecithin liposomes, the lipid phase transition is not significantly affected by valinomycin. However, in dimyristoylphosphatidylcholine liposomes, the phase transition is broadened by the addition of 1 mol% valinomycin even at low K⁺ concentrations. This indicates that the carrier interacts with the hydrophobic core of the bilayer. In addition, these experiments showed that the lipid phase transitions which are reflected by the methylene groups and the terminal methyl groups are nearly equivalent. Therefore a reevaluation of the assignment of the CH stretching bands seemed necessary. Our Raman spectroscopic investigation of ω-deuterated dipalmitoyl lecithin liposomes improves the assignment of CH stretch vibrations to methylene and methyl groups. The deuteration displaces the methyl group vibrations to the 2050–2250 cm^?1 region and produces gross intensity changes of the bands at 2883 and 2936 cm^?1. These changes lead to the conclusion that both bands arise from vibrations which can be attributed simultaneously to the methylene and methyl groups of the fatty acid chains. The displacement of the CH₃ group vibrations from their original positions enhances the intensity ratios (per centimeter), $\text{2883}\text{2847}$ and $\text{2936}\text{2847}$, for the CH₂- groups which are used to monitor the lipid phase transition, and implies that the contributions of the CH₃ groups to the phase transition curves are unimportant. Our finding that the -CD₃ groups reflect no phase transition supports this statement.     

Benzodiazepine binding in human brain: characterization using [3H]flunitrazepam.

[³H]Flunitrazepam was used to characterize benzodiazepine binding sites in human brain. The benzodiazepine binding sites exhibited high affinity, pharmacological specificity and saturability in their binding of [³H]flunitrazepam. The dissociation constant (K_D) of [³H]flunitrazepam binding was determined by three different methods and found to be in the range of 2–3 nM. The potency of several benzodiazepine analogs to inhibit specific [³H]-flunitrazepam binding $\text{in}$$\text{vitro}$ correlated well with their potency in several $\text{in}$$\text{vivo}$ human and animal tests. The density of [³H]-flunitrazepam binding sites was highest in the cerebrocortical and rhinencephalic areas, intermediate in the cerebellum, and lowest in the brain stem and commissural tracts.     

Pharmacologic identification of muscarinic receptors in the pylorus of the cat by binding of [3H]-quinuclidinyl benzilate

Muscarinic receptors in the smooth muscle of the cat pylorus (pyloric sphincter) were identified by binding of the ligand (±) [³H]-quinuclidinyl benzilate ([³H]-QNB). Receptor related binding of [³H]-QNB reached steady-state in thirty minutes at 37°C, was saturable, showed pharmacologic specificity and was stereoselective. An apparent equilibrium dissociation constant, K_D, of 1.9 ± 0.3 nM and maximum receptor concentration of 122 ± 13 femtomoles per mg of protein (means ± S.E.M.) were determined from Scatchard plots of [³H]-QNB binding. Hill coefficients of 0.99 and 1.01 indicated the absence of cooperative interactions. The muscarinic antagonists atropine and propantheline inhibited binding with IC₅₀ values in the nanomolar range, whereas bethanechol was over four orders of magnitude less potent. Noncholinergic agents had little or no effect on [³H]-QNB binding. The $\text{levo}$ isomer of QNB was about seventy times more effective at inhibiting binding than its $\text{dextro}$ isomer while $\text{dextro}$ benzetimide was greater than two thousand fold more active than $\text{levo}$ benzetimide. The isomers of another anticholinergic compound, tropicamide, also competed for [³H]-QNB binding sites in a stereoselective manner, the $\text{levo}$ isomer being eighty-five times more potent than the $\text{dextro}$ isomer.     

Demonstration of [3H] diazepam binding to benzodiazepine receptors in vivo.

Benzodiazepine receptors were labeled with [³H] diazepam following intravenous injection in rats. Binding of [³H] diazepam $\text{in}$$\text{vivo}$ to rat forebrain membranes was displaceable by co-injection of clonazepam or the pharmacologically active enantiomers of two benzodiazepines, B9 and B10, but was not displaced by equal doses of the pharmacologically in-active enantiomers. Binding of [³H] diazepam $\text{in}$$\text{vivo}$ was bserved in kidney, liver, and abdominal muscle, but was not stereospecifically diplaced in any peripheral tissue studied. The regional distribution of benzodiazepine receptors in brain was uneven, with specific [³H] diazepam binding being highest in the cerebral cortex and lowest in the ponsmedulla. Preliminary studies of the subcellular distribution of [³H] diazepam binding demonstrated highest specific binding to synaptosomal membranes. These data demonstrate the feasibility of labeling benzodiazepine receptors in rat brain $\text{in}$$\text{vivo}$.     

Partial purification and properties of a non-ligandin (3H) 3-methylcholanthrene binding protein from liver cytosol

(³H) 3-Methylcholanthrene binds $\text{in vivo}$ to a macromolecule in addition to the previously reported binding to ligandin in liver cytosol. The properties of this second molecule are identical to those of the glucocorticosteroid receptor (Binder II) through 400 fold purification over the cytosol proteins (elution position from DEAE-Sephadex A-50 columns, molecular weight by gel filtration and pI value by isoelectrofocusing). The carcinogen, probably a metabolite, binds very strongly or covalently to the macromolecule $\text{in vivo}$, but non-covalently $\text{in vitro}$ in the absence of microsomes. Large amounts of unlabeled carcinogen administered $\text{in vivo}$ do not compete significantly with subsequent (³H) dexamethasone binding to the hormone receptor fraction $\text{in vitro}$. Methylcholanthrene and dexamethasone do not compete for binding sites $\text{in vitro}$ on isolated unlabeled Binder II leading to the conclusion that the glucocorticosteroid receptor and the methylcholanthrene binding protein are distinct entities.     

Identification of diazepam binding in intack animals.

An $\text{in vivo}$ method for labeling specific benzodiazepine (BDZ) binding sites in brain was developed using intravenously injected [³H]diazepam. Labeling of these sites is blocked by pretreatment of animals with high doses of pharmacologically active BDZs (but not by an inactive BDZ). Using this $\text{in vivo}$ binding technique, specific BDZ binding is enhanced by pretreatment of rats with the GAB?A agonist muscimol or with amino-oxyacetic acid, which increases GABA levels in brain.     

Etomidate stereospecifically stimulates forebrain,but not cerebellar, 3H-diazepam binding

³H-Diazepam binding to a total particulate fraction of rat forebrain is enhanced by (+)-etomidate and GABA, but not by (?)-etomidate. The enhancement of ³H-diazepam binding by (+)-etomidate was due to a two-fold increase in binding affinity, the maximal number of sites remained unchanged. The degree of stimulation with (+)-etomidate was higher than that obtained with GABA. THIP did not stimulate ³H-diazepam binding to forebrain, and did not reverse the enhanced binding seen with (+)-etomidate or GABA. In a synaptosomal membrane preparation of rat cerebellum, unlike (+)-etomidate, GABA and muscimol produced a marked stimulation of ³H-diazepam binding. (+)-Etomidate did not inhibit ³-muscimol binding to GABA receptors, nor did it activate or inhibit other $\text{in vitro}$ receptor binding assays. The effects of (+)-etomidate on the benzodiazepine binding are different from those of gabamimetic drugs. It is proposed that like barbiturates, (+)-etomidate may affect benzodiazepine binding by interaction with the chloride ionophore which is coupled to the GABA-receptor.     

Effects of prolyl-leucyl-glycinamide and cyclo(leucyl-glycine) on morphine-induced antinociception and brain μ, δ and κ opiate receptors

The effects of prolyl-leucyl-glycinamide and cyclo (leucyl-glycine) on morphine-induced antinociception in mice and on $\text{in vitro}$ binding of ³H-ligands for opiate receptor subtypes (μ, δ and κ) the mouse brain homogenate were determined. Subcutaneous administration of either of the above peptides (1, 2, and 4 mg/kg) 10 min prior to the injection of morphine did not affect morphine-induced antinociception as evidenced by the identical ED₅₀ values of morphine in vehicle and peptide treated groups. The binding of ³H-dihydromorphine and ³H-naloxone ( μ receptors), ³HDAla²DLeu⁵-enkephalin (δ receptors), and ³H-ethylketocyclazocine (κ receptors) to opiate receptors in the mouse brain homogenate was also unaffected by both the peptides over a large concentration range. It is concluded that these peptides do not interact with brain opiate receptors.     

Effects of Δ9-tetrahydrocannabinol on neurotransmitter accumulation and release mechanisms in rat forebrain synaptosomes

The effects of (?)?Δ⁹-THC were studied on the release and accumulation of ³H5HT and ³HNE in a rat forebrain synaptosomal preparation. These studies were designed to evaluate the possible sites of action of Δ⁹-THC on these two processes. Δ⁹-THC inhibited the accumulation of ³H-leucine, ³HNE, and ³H5HT, as well as facilitated the release of the latter two amines (to a lesser degree), but had no effect on the release of ³H-leucine. Eighteen-hour pre-treatment with reserpine diminished the ability of Δ⁹-THC to induce release of ³H5HT, but had no effect on the $\text{in vitro}$ inhibition of synaptosomal uptake of this amine. Concentrations of Δ⁹-THC which blocked the uptake of ³H5HT also reduced the conversion of ³H5HT to ³H-5-hydroxy-3-indoleacetic acid. However, Δ⁹-THC, at concentrations which facilitated release of ³H5HT from preloaded synaptosomes, increased the amount of ³H5HIAA found in the medium. Taken together, these data suggest that Δ⁹-THC facilitates release from the synaptic vesicle and retards accumulation at the neuronal membrane.     

Demonstration of specific "high affinity" binding sites for [3H] imipramine on human platelets

High affinity and saturable binding sites for [³H] imipramine have been demonstrated on human platelet membranes. These binding sites appear to be specific for tricyclic antidepressants and their pharmacologically-active metabolites. In contrast, inactive tricyclic compounds such as the parent iminodibenzyl and iminostilbenes do not inhibit [³H] imipramine binding. The binding of [³H] imipramine to human platelets is of high affinity $\text{(}\text{Kd}\text{? 1.4}\text{nM}\text{)}$, saturable $\text{(}\text{Bmax}\text{? 625}\text{fmols/mg prot}\text{)}$, and sensitive to proteolytic degradation. The effects of various drugs and neurotransmitter agonists and antagonists suggests that these binding sites are pharmacologically distinct from the previously reported binding of tricyclic antidepressants to alpha-adrenergic, muscarinic-cholinergic, and histaminergic receptors. The binding characteristics of [³H] imipramine to platelets is similar to that in rat and human brain and may thus serve as a useful model in elucidating the pharmacological and physiological significance of these binding sites.     

Identification of alpha adrenoceptor subtypes in dog arteries by (3H) yohimbine and (3H) prazosin

Binding of the alpha adrenergic antagonists (³H) prazosin and (³H) yohimbine to membranes of dog arteries exhibit the characteristics expected of alpha adrenoceptors. Binding of both ligands is saturable with dissociation constants of 0.19nM and 1.15nM for (³H) prazosin and (³H) yohimbine respectively. A series of catecholamines inhibit binding of both ligands with a potency in the order $\text{epinephrine}\text{>}\text{norepinephrine}\text{a}\text{?}\text{isoproterenol}$, corresponding with the activity of these agents at alpha adrenoceptors in blood vessels. Competition for binding in both instances is stereoselective. ?-Phenylephrine has similar potencies in inhibiting (³H) prazosin and (³H) yohimbine specific binding whilst the imidazoline related partial alpha adrenergic agonists clonidine and guanfacine are more potent in inhibiting (³H) yohimbine specific binding. The affinity of prazosin for the (³H) yohimbine binding site is approximately 2500 times less than for the (³H) prazosin site whilst yohimbine is approximately 150 times more potent in inhibiting (³H) yohimbine than (³H) prazosin specific binding. Non-selective alpha adrenergic antagonists have similar affinities for both binding sites. The concentrations of (³H) yohimbine binding sites in different arteries vary about two fold whilst for (³H) prazosin the variation was about three fold. These results indicate that there are two discrete noradrenergic binding sites in the major arteries of dog which have binding properties expected of alpha₁ and alpha₂ adrenoceptors.     

Properties of [3H] diazepam binding to rat peritoneal mast cells

Benzodiazepine binding to rat peritoneal mast cells was investigated using [³H] diazepam as the radioactive probe. The specific binding of [³H] diazepam reaches equilibrium within 10–15 min, is saturable and is linear with cell number. Scatchard analysis of equilibrium binding indicates the existence of only one class of binding sites with a K_D = 90 ± 10 nM and B_max of 261 ± 60 fmoles/10⁶ cells. The binding of [³H] diazepam is temperature dependent, the highest amount is bound at 0°C and shows a pH-optimum between pH 6.8 – 7.4. The binding of [³H] diazepam is reversible with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.2 ± 0.2}\text{min.}$ Based on the relative potency of clonazepam and Ro5-4864 in displacing the specific [³H] diazepam binding, the binding sites in the mast cell are similar to those in the peripheral tissues like lung, liver, and kidney and are different from those in the brain. These data indicate that the mast cells have benzodiazepine binding sites which are of the peripheral type.     

DDT inhibition of Ca-Mg ATPase from peripheral nerves and muscles of lobster, Homarus americanus

Sensitivity of CaMg ATPase from axonic plasma membrane (APM) and sarcoplasmic reticulum (SR) of lobster, $\text{Homarus}\text{,}\text{americanus}$, to DDT was studied. The CaMg ATPase found in SR with the high Ca²⁺ affinity is sensitive to DDT while the portion of ATPase related to the low Ca²⁺ affinity site is not inhibited by DDT. Also, DDT is more inhibitory against the CaMg ATPase prepared from APM than the one obtained from SR. The relationship between inhibition of the CaMg ATPase by DDT in the axonic nerve membrane and $\text{in}\text{,}\text{vivo}$ poisoning symptoms of the nervous system is discussed.     

Catecholamine receptors and cyclic AMP formation in the central nervous system: effects of tetrahydroisoquinoline derivatives.

The ability of a series of tetrahydroisoquinoline (THIQ) alkaloids to inhibit the binding of radioligands to catecholamine receptors in the CNS has been examined. $\text{(+)}$ THP was the most potent inhibitor of [³H] dihydroalprenolol binding to β-adrenergic receptors and of [³H] haloperidol to dopaminergic receptors and was the least potent inhibitor of [³H] WB-4101 binding to α-adrenergic receptors. Other THIQ alkaloids examined such as salsoline, salsolinol, and reticuline were less potent than $\text{(+)}$ THP in inhibiting radioligand binding to β-adrenergic and dopaminergic receptors, and more potent than $\text{(+)}$ THP in inhibiting radioligand to α-adrenergic receptors. The marked potency of $\text{(+)}$ THP in inhibiting radioligand binding to β-adrenergic receptors (IC₅₀ ～ 10^?7 M) was confirmed by the potency of this compound in inhibiting (?) isoproternol elicited accumulations of cyclic AMP in brain slice preparations. These data indicate that, if formed $\text{in}$$\text{vivo}$ during alcohol consumption, THIQ derivatives such as THP may affect catecholamine neurons in the CNS.     

Conformational transitions in a Pro-Pro peptide on dissolution of single crystals

The peptide t-butyloxycarbonyl-α-aminoisobutyryl-L-prolyl-L-prolyl-N-methylamide has been shown to adopt an extended structure in the solid state. The Pro-Pro segment occurs in the poly-proline II conformation. On dissolution of single crystals at ～ 233°K, a single species corresponding to the all $\text{trans}$ peptide backbone is observed by 270 MHz ¹H NMR. On warming, $\text{trans}$ to $\text{cis}$ isomerization about the Pro-Pro bond is facilitated. Both $\text{cis'}$ (ψ ～?50°) and $\text{trans'}$ (ψ ～ 130°) rotamers about the Pro³ C^αCO bond are detectable in the Pro-Pro $\text{cis}$ conformer, at low temperature. These observations demonstrate unambiguously the large differences in the solid state and solution conformations of a Pro-Pro sequence.