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.     

Effects of the intracerebroventricular injection of antinociceptive doses of acetylcholine on the stereospecific binding of 3H-dihydromorphine

The antinociceptive effects of intraventricularly administered acetylcholine (ACh) and its congeners have been demonstrated by previous investigators. The opiate receptor binding concept was used in this study to investigate possible correlations between ACh antinociception and its effects on opiate stereospecific binding. ACh $\text{in vitro}$ decreased the stereospecific binding of ³H-dihydromorphine in mouse brain homogenates. Such decrease was also observed in the brain homogenates of mice which had been treated with ACh intracerebroventricularly (i.v.t.). The decrease in the stereospecific binding of ³H-dihydromorphine induced by (i.v.t.) acetylcholine was inhibited by naloxone, atropine, cyclazocine and pentazocine. The $\text{d}$-isomers of cyclazocine and pentazocine were more potent than the $\text{l}$-isomers in antagonizing the inhibitory effects of i.v.t. acetylcholine upon the stereospecific binding of ³H-dihydromorphine to mouse brain homogenates. The same stereospecificity of these two narcotic analgesics in blocking acetylcholine had been previously observed in the tail-flick test. It is suggested that the antinociceptive effects of acetylcholine are related to the inhibition of opiate stereospecific binding, the mechanism of which is yet to be understood.     

Naloxone antagonism of phenoxybenzamine antinociception in the mouse tail stimulation test.

Phenoxybenzamine was antinociceptive in the mouse tail electrical stimulation assay (ED50, 36.8 mg/kg) with a peak effect at $\text{2}\text{1}\text{2}$ hours after subcutaneous injection. Naloxone antagonized this antinociception action of phenoxybenzamine in a dose-related manner. Dose-ratio analysis of naloxone's antagonism of phenoxybenzamine antinociception gave a pA₂ value of 6.15, similar to that found for the benzomorphinan mixed agonist-antagonists. This is in agreement with the sodium response ratio found for phenoxybenzamine, 4.3, in $\text{in vitro}$ assays of phenoxybenzamine inhibition of ³H-naloxone binding to mouse brain homogenate (5). These findings suggest that phenoxybenzamine binds to the opiate receptor both $\text{in vivo}$ as well as $\text{in vitro}$ in a manner similar to the mixed agonist-antagonists.     

Involvement of spinal release of α-neo-endorphin on the antinociceptive effect of TAPA

The antinociceptive effect of i.t.-administered Tyr-d-Arg-Phe-β-Ala (TAPA), an N-terminal tetrapeptide analog of dermorphin, was characterized in ddY mice. In the mouse tail-flick test, TAPA administered i.t. produced a potent antinociception. The antinociception induced by TAPA was significantly attenuated by i.t. pretreatment with the κ-opioid receptor antagonist nor-binaltorphimine, as well as by the μ-opioid receptor antagonist β-funaltrexamine and the μ₁-opioid receptor antagonist naloxonazine. TAPA-induced antinociception was also significantly suppressed by co-administration of the μ₁-opioid receptor antagonist Tyr-d-Pro-Phe-Phe-NH₂ (d-Pro²-endomorphin-2) but not by co-administration of the μ₂-opioid receptor antagonists Tyr-d-Pro-Trp-Phe-NH₂ (d-Pro²-endomorphin-1) and Tyr-d-Pro-Trp-Gly-NH₂ (d-Pro²-Tyr-W-MIF-1). In CXBK mice whose μ₁-opioid receptors were naturally reduced, the antinociceptive effect of TAPA was markedly suppressed compared to the parental strain C57BL/6ByJ mice. Moreover, the antinociception induced by TAPA was significantly attenuated by i.t. pretreatment with antiserum against the endogenous κ-opioid peptide α-neo-endorphin but not antisera against other endogenous opioid peptides. In prodynorphin-deficient mice, the antinociceptive effect of TAPA was significantly reduced compared to wild-type mice. These results suggest that the spinal antinociception induced by TAPA is mediated in part through the release of α-neo-endorphin in the spinal cord via activation of spinal μ₁-opioid receptors.     

Pharmacokinetic and pharmacodynamic factors contributing to the convulsant action of β-carboline-3-carboxylic acid esters

Both the methyl ester of β-carboline-3-carboxylic acid and the 6, 7-dimethoxy-4-ethyl derivative of this compound are potent convulsants in rodents, while the ethyl ester of β-carboline-3-carboxylic acid does not cause convulsions, even when administered at very high doses. The rate of degradation of these compounds by rat plasma ($\text{in vitro}$) parallels their potencies as convulsants. In contrast, 3-carboethoxy-β-carboline was found to potently elicit tonic and clonic convulsions in the squirrel monkey ($\text{Saimiri sciureus}$). Furthermore, the rate of degradation of 3-carboethoxy-β-carboline in monkey plasma ($\text{in vitro}$) is negligible compared with rats. No significant differences were observed in either the potency or efficacy of GABA to inhibit [³H] β-carboethoxy-β-carboline binding in rat and monkey brain. These data strongly suggest that pharmacokinetic, as well as pharmacodynamic, factors may determine the pharmacologic profile of these β-carboline-3-carboxylic acid esters.     

Properties of a trypsin and chymotrypsin inhibitor secreted by larval Taenia pisiformis

Living metacestodes of Taenia pisiformis maintained in vitro discharge into the surrounding medium a protease inhibitor, which has been purified from the medium by affinity chromatography on bovine α-chymotrypsin immobilized to CNBr-activated Sepharose 4B. The purified inhibitor was shown to inactivate the hydrolysis of $\text{N-α-}\text{benzoyl}\text{-}\text{L}\text{-}\text{arginine}$ ethyl ester and $\text{N-}\text{benzoyl}\text{-}\text{L}\text{-}\text{tyrosine}$ ethyl ester, respectively, by trypsin and chymotrypsin of bovine, rabbit and dog origin, and also the hydrolysis of casein by both bovine trypsin and bovine α and β chymotrypsins, but it did not affect the enzymic activity of subtilisin, elastase, collagenase, pepsin, rennin and papain. The inhibitor withstood heating at 100°C for up to 30 min, was stable in the pH range of 1.5–8.0, was unaffected by 8 M-urea or 0.2 M-2-mercaptoethanol, and had a molecular weight of about 7000 as calculated from its gel chromatographic behaviour. The inhibitor specifically inhibits either trypsin or chymotrypsin with the formation of stable enzyme inhibitor complexes that are not dissociated by 4 M-KCl. Inhibition of trypsin and chymotrypsin is non-competitive and is linear with inhibitor concentration up to 70–80% inhibition. Inhibitory activities toward both enzymes are functions of the same binding site of the inhibitor molecule. Complex formation between the inhibitor and the enzymes is timedependent; it requires 3–4 min for completion.     

Effect of Nipecotic Acid, a γ-Aminobutyric Acid Transport Inhibitor, on the Turnover and Release of γ-Aminobutyric Acid in Rat Cortical Slices

Abstract: To see the effect of a γ-aminobutyric acid GABA uptake inhibitor on the efflux and content of endogenous and labeled GABA, rat cortical slices were first labeled with [³H]GABA and then superfused in the absence or presence of 1 mM nipecotic acid. Endogenous GABA released or remaining in the slices was measured with high performance liquid chromatography, which was also used to separate [³H]GABA from its metabolites. In the presence of 3 mM K⁺, nipecotic acid released both endogenous and [³H]GABA, with a specific activity four to five times as high as that present in the slices. The release of labeled metabolite(s) of [³H]GABA was also increased by nipecotic acid. The release of endogenous GABA evoked by 50 mM K⁺ was enhanced fourfold by nipecotic acid but that of [³H]GABA was only doubled when expressed as fractional release. In a medium containing no Ca²⁺ and 10 mM Mg²⁺, the release evoked by 50 mMK⁺ was nearly suppressed in either the absence or the presence of nipecotic acid. In the absence of nipecotic acid electrical stimulation (bursts of 64 Hz) was ineffective in evoking release of either endogenous or [³H]GABA, but in the presence of nipecotic acid it increased the efflux of endogenous GABA threefold, while having much less effect on that of [³H]GABA. Tetrodotoxin (TTX) abolished the effect of electrical stimulation. Both high K⁺ and electrical stimulation increased the amount of endogenous GABA remaining in the slices, and this increase was reduced by omission of Ca²⁺ or by TTX. The results suggest that uptake of GABA released through depolarization is of major importance in removing GABA from extracellular spaces, but the enhancement of spontaneous release by nipecotic acid may involve intracellular heteroexchange. Depolarization in the presence of Ca²⁺ leads to an increased synthesis of GABA, in excess of its release, but the role of this excess GABA remains to be established.     

L-NAME causes antinociception by stimulation of the arginine-NO-cGMP pathway

NG-nitro-L-arginine methyl ester (L-NAME) has been used extensively as a paradigmatic inhibitor of NO synthase and has been shown to cause antinociception in several experimental models. We describe here how L-NAME produced a dose-dependent antinociceptive effect when injected intraperitoneally in the mouse after acetic acid induced writhings, or intraplantarly in the rat paw pressure hyperalgesia induced by carrageenin or prostaglandin E2. In contrast another NO synthase inhibitor, NG-monomethyl-L-arginine (L-NMMA), had no significant effect per se but inhibited L-NAME systemic induced antinociception in mice and local induced antinociception in the rat paw hyperalgesia test. D-NAME had no antinociceptive effect upon carrageenin-induced hyperalgesia. Pretreatment of the paws with two inhibitors of guanylate cyclase, methylene blue (MB) and 1H-:[1,2,4]-oxadiazolo-:[4,3-a] quinoxalin-1-one (ODQ) abolished the antinociceptive effect of L-NAME. L-Arginine and the cGMP phosphodiesterase inhibitor, MY 5445 significantly enhanced the L-NAME antinociceptive effect. The central antinociceptive effect of L-NAME was blocked by co-administration of L-NMMA, ODQ and MB. The present series of experiments shows that L-NAME, but not L-NMMA, has an antinociceptive effect. It can be suggested that L-NAME causes the antinociceptive effect by stimulation of the arginine/ NO/ cGMP pathway, since the antinociceptive effect of L-NAME can be antagonized by L-NMMA and abolished by the guanylate cyclase inhibitors (MB and ODQ). In addition, the NO synthase substrate, L-arginine and the cGMP phosphodiesterase inhibitor, MY5445 were seen to potentiate the effects of L-NAME. Thus, L-NAME used alone, has limitations as a specific inhibitor of the arginine-NO-cGMP pathway and may therefore be a poor pharmacological tool for use in characterising participation in pathophysiological processes.     

Semisynthetic ‘acid-esterase’: Conformational modification of ribonuclease

Bovine pancreatic ribonuclease has been modified by exposure to acidic conditions, addition of indole propionic acid and crosslinking with glutaraldehyde. The ‘acid-esterase’ generated was purified up to 100-fold by ammonium sulphate fractionation and gel filtration on Biogel P-30. The partially purified acid-esterase hydrolysed tryptophan ethyl ester (TrEE) and $\text{N-}\text{benzoyl}\text{-}\text{l}\text{-}\text{arginine}$ ethyl ester (BAEE) effectively at pH 6.0–6.3, but it had very little activity towards glycine ethyl ester and lysine ethyl ester. Hydrolysis of TrEE was competitively inhibited by tryptophan. The acid-esterase exhibited amidase activity towards benzoyl-l-arginine p-nitroanilide.     

Dibutyryl cGMP: inhibitor of the effect of cholecystokinin and gastrin on the guinea pig gallbladder in vitro

N², O²-di-butyryl guanosine 3′:5′ monophosphate (Bt₂ cGMP), a known competitive and selective inhibitor of the effect of cholecystokinin on the pancreatic acinar cells $\text{in}$$\text{vitro}$ was tested for its effect on the guinea pig gallbladder $\text{in}$$\text{vitro}$. Bt₂ cGMP inhibited competitively the contractile effect of cholecystokinin octapeptide, and also inhibited the contraction induced by sulfated gastrin-17. Bt₂ cGMP failed to inhibit the contraction induced by bombesin, acetylcholine or histamine. The 8-bromo derivative of cGMP and the dibutyryl derivative of cAMP did not affect contraction stimulated by cholecystokinin octapeptide. Since it is specific for gastrincholecystokinin peptides, and not restricted to the pancreas, Bt₂ cGMP could be used to recognize the action of these peptides.     

Atropine lowers blood pressure in normotensive rats through blockade of α-adrenergic receptors

Atropine sulfate elicited a dose-dependent decrease in blood pressure in normotensive rats at doses higher than needed to cause muscarinic blockade. This hypotensive effect was not altered by pretreatment with ganglionic or β-adrenergic blockers, but was fully abolished by α-adrenergic blockers. In addition, atropine inhibited the pressor response to α-agonists in a dose-dependent manner. The time course for hypotension and α-blockade were the same (onset < 1 minute; duration < 20 minutes). $\text{In vitro}$, atropine was found to be 200 times more potent in displacing the α₁-adrenergic receptor ligand ([³H] WB-4101) than the α₂-ligand ([³H] clonidine). Thus the observed hypotensive effect is apparently due to α-blockade as demonstrated $\text{in vivo}$ and $\text{in vitro}$.     

Role of nitric oxide/cyclic GMP/K(+) channel pathways in the antinociceptive effect caused by 2,3-bis(mesitylseleno)propenol

The present study examined the antinociceptive effects induced by 2,3-bis(mesitylseleno)propenol, a bis-selenide alkene derivate, given orally, in chemical models of pain in rats and mice. Selenide administered orally (p.o.) into the rats caused antinociception against the first and second phases of the formalin test, with mean ID(50) values of 28.17 and 39.68 mg/kg, respectively. The antinociceptive effect caused by selenide (50 mg/kg, p.o.) on the formalin test was reversed by pretreatment with N(G)-L-nitro-arginine methyl ester (L-NAME, a nitric oxide (NO) synthase inhibitor), methylene blue (a non-specific NO/guanylyl cyclase inhibitor) and glibenclamide (an ATP-sensitive K(+) channel inhibitor), but not by atropine (a muscarinic antagonist). Given orally selenide in mice produced an inhibition of glutamate-, histamine- and compound 48/80-induced nociception with mean ID(50) values of 27.58, 36.18 and 44.53 mg/kg, respectively. Moreover, oral treatment with selenide in mice decreased licking -- induced by serotonin (mean ID(50) value of >50 mg/kg). The data show that selenide exerts pronounced systemic antinociception in chemical (formalin, glutamate, histamine, compound 48/80 and serotonin-induced pain) models of nociception. Taken together, these results suggest that the antinociceptive effect of selenide on the formalin test involves the participation of nitric oxide/cyclic GMP/K(+) channel pathways in rats.     

Kinetics of in vivo binding of antagonist to muscarinic cholinergic receptor in the human heart studied by positron emission tomography

Positron Emission Tomography (PET) was used to analyse $\text{in vivo}$ antagonist binding to human myocardial muscarinic cholinergic receptor. The methiodide salt of the muscarinic antagonist, quinuclidinyl benzilate (MQNB), was labeled with the positron emitter, Carbon-11, and injected intravenously to 8 normal subjects. ¹¹C-MONB concentration was determined $\text{in vivo}$ in the ventricular septum from 40 cross-sectional images acquired at the same transverse level over a period of 70 minutes. In 4 subjects, various amounts of unlabeled atropine were rapidly injected at 20 minutes to study whether atropine competitively inhibited MQNB.The kinetics of binding of ¹¹C-MQNB were not the same $\text{in vivo}$ and $\text{in vitro}$. The apparent dissociation rate of ¹¹C-MQNB $\text{in vivo}$ was much slower (by 1 to 2 orders of magnitude) than that observed $\text{in vitro}$ with ³H-QNB. After atropine injection, ¹¹C-MNQB dissociated from its binding sites at a rate that apparently depended on the amount of atropine present. ¹¹C-MQNB kinetics were analysed with a mathematical model which assumes the existence of a boundary layer containing free ligand in the vicinity of the binding sites. The dissociation rate of the radioligand depends on the probability of its rebinding to a free receptor site.     

The reduction of vinyl side-chains of Mg-protoporphyrin IX monomethyl ester in vitro

Portions of crude homogenates of etiolated wheat seedlings incubated with Mg-protoporphyrin IX and $\text{S-}\text{adenosyl}\text{-}\text{L}\text{-}\text{methionine}$ and then added to other portions of the same crude homogenates that were pretreated with [1-³H]ethanol and yeast alcohol dehydrogenase provided, after a short reaction period, ³H-labeled Mg-protoporphyrin IX monomethyl ester. The ³H-labeled Mg-protoporphyrin IX monomethyl ester thus obtained was shown to contain the ³H in one reduced (to ethyl) vinyl side-chain. Subsequently, ³H-labeled Mg-monoethyl-(monodivinyl)-protoporphyrin IX monomethyl ester was obtained when Mg-protoporphyrin IX monomethyl ester and [³H]NADH were added to dialyzed crude homogenates of etiolated wheat seedlings. Insignificant amounts of ³H were incorporated into poprhyrin substrates when Mg-2,4-divinylpheoporphyrin a₅ or [³H]NADPH were substituted in reaction mixtures for Mg-protoporphyrin IX monomethyl ester or [³H]NADPH, respectively. The results of these and further experiments suggest that an NADPH-dependent enzyme in the crude homogenates of etiolated wheat seedlings was capable of catalyzing the reduction to ethyl of one vinyl side-chain of Mg-protoporphyrin IX monomethyl ester. These findings suggest that the 4-vinyl side-chain reductive reaction likely occurs after the biosynthesis IX monomethyl ester, but before isocyclic ring formation in the pathway to chlorophyll a.     

Passive avoidance behavior: Opposite effects of oxytocin analogs with agonist and antagonist properties

Deamino-6-carba-oxytoxin (dC6O), a potent oxytocin analog considered to be resistant to some of the physiologically significant enzymic systems, and $\text{N-α-}\text{acetyl}\text{-[2-O-}\text{methyltyrosine}\text{]}\text{oxytocin}$ (AMTO), an analog acting as a competitive inhibitor of oxytocin on the rat uterus, were studied in rats trained in a passive avoidance task.Subcutanaeous administartion of dC6O (5–50 gmg·kg^?1) during different phases of the passive avoidance learning paradigm attenuated avoidance latencies; the results indicated that the drug induced state-dependent learning.AMTO (5–20 gmg·kg^?1) enhanced avoidance latencies when administered subcutaneously before training trials and/or before retention test trials. This effect occured in both males and females. The analogs did not influence exploratory behavior in open field.The results suggest that oxytoxin, in contrast to vasopressin, may impair memory processes. However, both analogs failed to influence the passive avoidance response when administered after training. This finding indicates that dC6O and AMTO did not influence the mechanism of memory consolidation whereas vasopressin and oxytoxin had a marked effect.     

Effects of γ-aminobutyric acid on 33Pi incorporation into rat brain phospholipids in vivo

The effects of γ-aminobutyric acid (GABA), bicuculline and strychnine on the incorporation $\text{in vivo}$ of ³³Pi into phospholipids of rat brain were studied at 10 and 30 minutes after intracisternal injection of the radionuclide. GABA inhibited labeling of phospholipids in the three brain regions studied at both times. Bicuculline by itself had no significant effect on ³³Pi incorporation, but totally blocked the inhibitory effect of GABA in all three brain regions. Strychnine by itself inhibited phospholipid labeling in the brain stem and forebrain, had no significant effect on GABA inhibition of ³³Pi incorporation in the cerebellum and forebrain, and partially blocked the GABA effect in the brain stem. GABA inhibited ³³Pi incorporation into phosphatidic acid, phosphatidylinositol, phosphatidyl choline and phosphatidyl ethanolamine but had no effect on phosphatidyl serine. The data suggest that the inhibitory effects of GABA on CNS phospholipid labeling are mediated specifically through GABA receptor sites.     

Metformin and phenformin block the peripheral antinociception induced by diclofenac and indomethacin on the formalin test

AimsRecent evidence has shown that systemic administration of sulfonylureas and biguanides block the diclofenac-induced antinociception, but not the effect produced by indomethacin. However, there are no reports about the peripheral interaction between analgesics and the biguanides metformin and phenformin. Therefore, this work was undertaken to determine whether glibenclamide and glipizide and the biguanides metformin and phenformin have any effect on the peripheral antinociception induced by diclofenac and indomethacin.Main methodsDiclofenac and indomethacin were administered locally in the formalin-injured rat paw, and the antinociceptive effect was evaluated using the 1% formalin test. To determine whether peripheral antinociception induced by diclofenac or indomethacin was mediated by either the ATP-sensitive K⁺ channels or biguanides-induced mechanisms, the effect of pretreatment with the appropriates vehicles or glibenclamide, glipizide, metformin and phenformin on the antinociceptive effect induced by local peripheral diclofenac and indomethacin was assessed.Key findingsLocal peripheral injections of diclofenac (50–200 μg/paw) and indomethacin (200–800 μg/paw) produced a dose-dependent antinociception during the second phase of the test. Local pretreatment with glibenclamide, glipizide, metformin and phenformin blocked the diclofenac-induced antinociception. On the other hand, the pretreatment with glibenclamide and glipizide did not prevent the local antinociception produced by indomethacin. Nonetheless, metformin and phenformin reversed the local antinociception induced by indomethacin.SignificanceData suggest that diclofenac could activate the K⁺ channels and biguanides-dependent mechanisms to produce its peripheral antinociceptive effects in the formalin test. Likewise, a biguanides-dependent mechanism could be activated by indomethacin consecutively to generate its peripheral antinociceptive effect.     

Effects of angiotensin I and II and their interactions with some prostanoids in perfused human umbilical arteries

In perfused human umbilical arteries both angiotensin I and II induced vasoconstriction with a monophasic response. Angiotensin I and II induced vasoconstrictions at doses ≥10^?8M and 10^?9 M respectively. Captopril inhibited the angiotensin I response while the angiotensin II receptor blocker Sar¹-Ala⁸ AII inhibited the effect of both angiotensins. PGI₂ attenuated the angiotensin II response in a dose dependent pattern. PGE₂ and PGF_2α in concentrations below the critical levels for creating pressure responses $\text{per se}$, also attenuated the angiotensin II response. The cyclooxygenase inhibitor indomethacin potentiated the angiotensin II response indicating that endogenous production of prostanoids is of importance in the modulation of angiotensin effects.     

Effect of phencyclidine on [3H]QNB binding

Intraperitoneal injection of phencyclidine before intravenous injection of [³H] Quinuclidinyl benzilate (QNE, 1.6 μg/kg) significantly increased the amount of radioactivity found in the brains of female C57BL/6J mice one hour after the ³H-QNB administration. This effect was found in hypothalamus, cortex, hippocampus and striatum and was decreased by pretreatment of the animal with atropine. The magnitude of the enhancement varied as a function of dose but did not change across the time span studied. These data are in contrast to our findings and those of others of inhibition of the specific binding of ³H-QNB to muscarinic cholinergic receptors by PCP $\text{in vitro}$. When atropine or PCP was administered $\text{in vivo}$ and the tissue later analyzed $\text{in vitro}$, no effects of the drugs were observed on ³H-QNB binding. The reasons for the differences remain a matter of speculation.     

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.