Endogenous regulators of benzodiazepine recognition sites

The method used to prepare crude synaptic membranes (CSMs) from rat brain affects the results obtained for the binding characteristics of ³H-diazepam and the GABA-induced stimulation of ³H-diazepam to CSM. In freshly prepared membranes (rich in GABA and other endogenous inhibitory factors), the K_D for ³H-diazepam is approximately 10 nM and the threshold dose of GABA needed to stimulate this binding is approximately 10^?5M. Removal of GABA resulted in an increase in the K_D values for ³H-diazepam binding. In contrast removal of endogenous inhibitory factors (by treatment of the membranes with Triton X-100) resulted in a decrease of the K_D values. In the Tritron X-100 treated membranes the threshold dose of GABA (10^?8M) required to stimulate ³H-diazepam binding is in the range of the high affinity component of ³H-GABA binding. Addition of crude preparations of endogenous inhibitor to these membranes increased the K_D of ³H-diazepam and inhibited the GABA-induced stimulation of ³H-diazepam binding.     

Isolation and chemical characterization of agelaiatoxin8 (AvTx8) from Agelaia vicina wasp venom and its biological effects on GABA neurotransmission

Arthropod venoms are sources of molecules that may be useful tools to investigate molecular mechanisms of putative new medicines and laboratory drugs. Here we show the effects of the compound agelaiatoxin‐8 (AVTx8), isolated from Agelaia vicina venom, on γ‐aminobutyric acid (GABA) neurotransmission in rat brain synaptosomes. Analysis reveals that AvTx8 is composed by 14 amino acid residues with a molecular weight (MW) of 1567 Da. AvTx8 increased GABA release and inhibited GABA uptake in synaptosomes from rat cerebral cortex. AvTx8 inhibited GABA uptake and increased GABA release in the presence of Ca⁺, Na⁺, and K⁺ channel blockers, suggesting that it acts directly on GABA transporters. In addition, AvTx8 significantly decreases GABA binding in synaptic membranes from rat brain cortex, suggesting that it also modulates the activity of GABA receptors. Moreover, AvTx8 decreased GAT‐1– and GAT‐3–mediated GABA uptake in transfected COS‐7 cells. Accordingly, we suggest that AvTx8 modulates GABA neurotransmission and might provide a novel entry point for identifying a new class of GABA‐modulating neuroprotective drugs.     

Anticonvulsant Activity of Muscimol and γ-Aminobutyric Acid Against Pyridoxal Phosphate-Induced Epileptic Seizures

The intracerebroventricular injection of pyridoxal phosphate (PLP, 0.125-1.25 μmol/rat) causes epileptic seizures (4 min → 1 min) that are preventable or reversible by GABA (1 μmol/rat), by muscimol (O.025 μmol/rat), or by diazepam (1.75 μmol/rat). At the peak of PLP-induced convulsions, the activities of GAD and GABA-T in 14 regions of rat brain remained unaltered, whereas the concentrations of PLP remained elevated. The PLP-induced convulsion was blocked by DABA (10 μmol/rat) but was not altered by β-alanine (50 μmol/rat). The previous in vitro studies have shown that PLP increases the uptake of [³H]GABA into synaptosomes and inhibits the binding of [³H]GABA to synaptic membranes. These data suggest that PLP-induced convulsion is due to reduced availability of GABA to its recognition sites, rather than to alteration in the activity of GABA metabolizing enzymes, or unavailability of PLP as a coenzyme for GAD and GABA-T. Since the duration of PLP-induced epileptic seizures is short and can be prevented by GABA agonists, PLP may be used as a tool to study the nature of GABA-mediated neuroinhibition and the properties of GABA receptor sites.     

Glutamic Acid and γ-Aminobutyric Acid Modulate Each Other's Release Through Heterocarriers Sited on the Axon Terminals of Rat Brain

Abstract: The effects of γ-aminobutyric acid (GABA) on the spontaneous release of endogenous glutamic acid (Glu) or aspartic acid (Asp) and the effects of Glu on the release of endogenous GABA or [³H]GABA were studied in superfused rat cerebral cortex synaptosomes. GABA increased the outflow of Glu (EC₅₀17.2 μM) and Asp (EC₅₀ 18.4 μM). GABA was not antagonized by bicuculline or picrotoxin. Neither muscimol nor (-)-baclofen mimicked GABA. The effects of GABA were prevented by GABA uptake inhibitors and were Na⁺ dependent. Glu enhanced the release of [³H]GABA (EC₅₀ 11.5 μM) from cortical synaptosomes. Glu was not mimicked by the glutamate receptor agonists N-methyl-d -aspartic, kainic, or quisqualic acid. The Glu effect was decreased by the Glu uptake inhibitor D-threo-hydroxyaspartic acid (THA) and it was Na⁺ sensitive. Similarly to Glu, D-Asp increased [³H]GABA release (EC₅₀ 9.9 μM), an effect blocked by THA. Glu also increased the release of endogenous GABA from cortex synaptosomes. In this case the effect was in part blocked by the (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist 6-cyano-7-nitroquinoxaiine-2, 3-dione, whereas the 6-cyano-7-nitroquinoxaline- 2, 3-dione-insensitive portion of the effect was prevented by THA. GABA increased the [³H]D-Asp outflow (EC₅₀ 13.7 μM) from hippocampal synaptosomes in a muscimol-, (-)- baclofen-, bicuculline-, and picrotoxin-insensitive manner. The GABA effect was abolished by blocking GABA uptake and was Na⁺ dependent. Glu increased the release of [³H]- GABA from hippocampal synaptosomes (EC₅₀ 7.1 μM) in an N-methyl-d -aspartic acid-, kainic acid-, or quisqualic acid-insensitive way. The effect of Glu was prevented by THA and was Na⁺ dependent. As in the cortex, the effect of Glu was mimicked by D-Asp in a THA-sensitive manner. It is proposed that high-affinity GABA or Glu heterocarriers are sited respectively on glutamatergic or GA- BAergic nerve terminals in rat cerebral cortex and hippocampus. The uptake of GABA may modulate Glu and Asp release, whereas the uptake of Glu may modulate the release of GABA. The existence of these heterocarriers is in keeping with the reported colocalization of GABA and Glu in some cortical and hippocampal neurons. Preliminary data suggest that these mechanisms may also be present in rat cerebellum and spinal cord.     

A modulating role of mercurials-sensitive sulfhydryl groups in synaptic GABA receptor binding

The specific binding of GABA (γ-aminobutyric acid) agonist ³H-muscimol, to synaptic membranes from the rat brain showed a significant increase, when the membranous preparations were treated with a low concentration (10^?4–10^?5M) of mercurial sulfhydryl reagents such as p-chloromercuribenzoate and mercuric chloride. This activation in GABA receptor binding was bicuculline-sensitive, and was partially restored by subsequent treatments with 10 mM cysteine, penicillamine, or mercaptoethanol. Scatchard analysis of the binding revealed that this activation was due to the increase in the affinity of both high and low affinity bindings sites but not in the B_max values. On the other hand, the treatment of synaptic membranes with hydrophilic sulfhydryl reagents such as N-ethylmaleimide and iodoacetate had no effect on the binding. These hydrophilic sulfhydryl reagents, however, induced an increase of the binding following the pretreatment of synaptic membranes with 0.01% Triton X-100 or 0.5 U/mg prot. of phospholipase A₂ (EC 3.1.1.4.). These results suggest that mercurials-sensitive sulfhydryl groups, which are normally masked by membrane lipids, may play a modulating role in GABA receptor binding at central synapses.     

gamma-Aminobutyric acid receptors in cerebellar membranes of rat brain after a treatment with Triton X-100.

Abstract— The treatment of cerebellar membranes of rat brain with a low concentration of Triton X-100 followed by sufficient washing results in an increase of the Na⁺-independent binding of [³H]GABA and a total loss of the Na ⁺-dependent binding of [³H]GABA. The Na⁺-independent binding of [³H]GABA was more abundant in membranes of cerebellum than in membranes of other rat brain regions and mainly localized in the synaptic membrane fraction of a cerebellar homogenate. In the Triton-treated membranes, the Na⁺-independent binding of [³H]GABA was a saturable process, which could be resolved into two components, a high and a low affinity component with dissociation constants of 4.5 and 30 nm , respectively. The neurophysiological agonists, muscimol, GABA, and imidazole acetic acid, and the antagonist, bicuculline, inhibited the high affinity Na⁺-independent binding of [³H]GABA by 50% at 0.003, 0.012, 0.3 and 10 μm respectively. These data suggest that the Na⁺-independent binding of [³H]GABA in the Triton-treated cerebellar membranes represents the synaptic receptors of GABA. It is emphasized that extensive washing of the membranes after a Triton treatment is necessary in order to detect the high affinity Na⁺-independent binding of [³H]GABA.     

Na+-independent binding of GABA to the triton X-100 treated synaptic membranes from cerebellum of rat brain

The treatment of the membranes from cerebellum of rat brain with 0.5% Triton X-100 increases both the affinity and the density of the Na⁺-independent binding sites for ³H-GABA (γ-aminobutyric acid) from the values obtained from membranes of rat brain after an extensive freezing and thawing treatment (Young $\text{et al.}$, 1976). Upon repeated washings of the Triton-treated membranes, the binding of ³H-GABA is further increased and follows biphasic kinetics which indicates two binding components having dissociation constants of 5.9 and 27 nM and densities of 1.35 and 3.9 pmole/mg protein, respectively. GABA agonist, imidazoleacetic acid, and the GABA antagonists, bicuculline and d-tubocurarine, inhibit 50% of ³H-GABA binding at 1, 47 and 85 μM concentrations (IC₅₀ values), respectively. The IC₅₀ values for these compounds are unchanged by Na⁺. Thus, the Na⁺-independent binding of ³H-GABA to the Triton-treated membranes may represent binding to the synaptic GABA receptors.     

The GABA/benzodiazepine receptor complex in the nervous system of a hypertensive strain of rat

-Aminobutyric acid (GABA) has been implicated in the development of hypertension and in the regulation of blood pressure. The spontaneously hypertensive rat (SHR) offers an opportunity to explore the role of central GABA and other neurotransmitters in the genesis of high blood pressure. The receptor binding of [³H]GABA, [³H]flunitrazepam, and [³H]glutamate to synaptic membranes from the cerebral cortex and cerebellum of SHR rats were measured in animals of various ages. No significant differences between the SHR and a normotensive control strain of rats were found for any of the assays. The results indicate that in this model of hypertension, neither GABA nor glutamate function are involved, at least not in the cerebral cortex or cerebellum.     

Enhancement of the binding of 3H-diazepam to rat brain membranes in vitro by SQ 20009, A novel anxiolytic, gamma-aminobutyric acid (GABA) and muscimol.

SQ 20009, a compound with anxiolytic-like activity, was found to cause an enhancement of the binding of ³H-diazepam to rat brain membranes in a concentration-dependent manner with inhibition of binding occuring at 10^?4 M. Both GABA and the GABA agonist, muscimol, were also found to enhance ³H-diazepam binding while the GABA antagonist (+)-bicuculline decreased binding. The effects of SQ 20009, the GABA agonists and (+)-bicuculline were reflected as a change in the affinity rather than number of binding sites. The effects of SQ 20009 and GABA and muscimol are discussed in terms of an interaction with an endogenous diazepam-like factor.     

ISOLATION OF HYDROPHOBIC PROTEINS BINDING AMINO ACIDS: γ-AMINOBUTYRIC ACID BINDING IN THE RAT CEREBRAL CORTEX

—The binding of [¹⁴C]GABA to nerve-ending membranes isolated from rat cerebral cortex follows a hyperbolic curve saturating at 0·4pmol/μg protein. This binding is about 60% inhibited by chloropromazine, and about 40%, inhibited by bicuculline. A hydrophobic protein fraction binding [¹⁴C]GABA was separated from the total. lipid extract of nerve-ending membranes. The binding follows a hyperbolic curve that saturates at 10·5 pmol of [¹⁴C]GABA/μg of protein, with an apparent K_d= 30 μm . The binding is competitively inhibited by bicuculline with a K_i= 273 μm . These results are compared with those previously obtained on a GABA binding protein from crustacean muscle.     

Substrate Specificity of [3H] Muscimol Binding to a Particulate Fraction of a Neuron-enriched Culture of Embryonic Rat Brain

Abstract: The effects of some GABA analogues and some drugs on the binding of [³H]muscimol (3.08 nM) to thoroughly washed subcellular particles prepared from a neuron-enriched culture of embryonic rat brain were examined using Na+-free Tris-citrate medium and a centrifugation method. Competition for [³H]muscimol binding sites by excess(10^?5 M) unlabelled GABA provided estimates of “specific” binding. In accord with in vivo neuropharmacological studies on GABA receptors and with in vitro studies on cerebral membrane preparations, [³H]muscimol binding was potently inhibited by muscimol itself (IC₅₀, 2.5 nM), GABA (1C₅₀, 43 nM), isoguvacine (IC₅₀, 61 nM), and 3-aminopropanesulphonic acid (IC₅₀, 160 nM), and less potently inhibited by the GABA antagonist bicuculline methobromide (IC₅₀, 800 nM). δ- Aminovaleric acid (IC₅₀, 2.6 μM), the glycinelp-alanine antagonist strychnine (IC₅₀, 6.6 μM), and the predominantly glial GABA uptake inhibitors β-alanine (IC₅₀, 23 μM) and p-proline (IC₅₀, 66 μM) also inhibited [³H]muscimol binding. Other inhibitors of Na+-dependent GABA uptake, (±)-nipecotic acid, L- 2,4-diaminobutyric acid, and guvacine, as well as picrotoxinin, were relatively inactive as inhibitors of [³H]muscimol binding (IC₅₀≥ 1 mM). In addition to revealing that GABA receptors are present on neuronal membranes before the formation of most synapses, this binding of [³H]muscimol that occurs to neuronal, but not to glial, membranes might be useful as a “neuronal marker” and for the further characterization and isolation of GABA receptors.     

COMPARISON OF THE BINDING’ OF β-ALANINE AND y-AMINOBUTYRIC ACID IN SYNAPTOSOMAL-MITOCHONDRIAL FRACTIONS OF RAT BRAIN1

Abstract— Na⁺-dependent ‘binding’ of β-alanine and GABA was examined with synaptosomal-mitochondrial fractions of rat brain incubated for 10 min at 0°C. GABA was bound to a much greater extent than β-alanine to particles of cerebral cortex, whole cerebellum and brain stem. For cerebral cortex, the binding capacity (B_max) for GABA was about 18 limes greater than that for β-alanine. and the affinity of the particles for GABA was about 2′ times greater than for β-alanine. The order of potency of GABA binding to brain regions was cerebral cortex > cerebellum > brain stem, whereas that for β-alanine was the reverse. If the binding of β-alanine is taken to indicate the glial component of the Na⁺-dependent binding process for GABA, then most of the GABA was bound to neuronal elements under the conditions employed.     

THE UPTAKE OF [3H]GABA BY SLICES OF RAT CEREBRAL CORTEX

—A rapid accumulation of [³H]GABA occurs in slices of rat cerebral cortex incubated at 25° or 37° in a medium containing [³H]GABA. Tissue medium ratios of almost 100:1 are attained after a 60 min incubation at 25°. At the same temperature no labelled metabolites of GABA were found in the tissue or the medium. The process responsible for [³H]GABA uptake has many of the properties of an active transport mechanism: it is temperature sensitive, requires the presence of sodium ions in the external medium, is inhibited by dinitrophenol and ouabain, and shows saturation kinetics. The estimated K_m value for GABA is 2·2 × 10^?5m , and V_max is 0·115 μmoles/min/g cortex. There is only negligible efflux of the accumulated [³H]GABA when cortical slices are exposed to a GABA-free medium. [³H]GABA uptake was not affected by the presence of large molar excesses of glycine, l -glutamic acid, l -aspartic acid, or β-aminobutyrate, but was inhibited in the presence of l -alanine, l -histidine, β-hydroxy-GABA and β-guanidinopropionate. It is suggested that the GABA uptake system may represent a possible mechanism for the inactivation of GABA or some related substance at inhibitory synapses in the cortex.     

Melatonin reverses pinealectomy-induced decrease of benzodiazepine binding in rat cerebral cortex

Pinealectomy of rats resulted in significant depression of benzodiazepine receptors (assessed by [³H]flunitrazepam binding) in cerebral cortex 3–14 days after surgery without affecting their affinity significantly. A single s.c. injection of melatonin (800 μg/kg body wt) restored the depressed brain benzodiazepine receptor sites. Single melatonin injections (up to 1600 μg/kg) to intact rats did not affect brain benzodiazepine binding when injected at either morning or evening hours. Daily melatonin treatment to intact rats for 5 days augmented benzodiazepine receptor density in brain (morning injections) or its dissociation constant (evening injections). Melatonin added in vitro to rat cerebral cortex membranes only slightly depressed [³H]flunitrazepam binding at 100 μM concentrations. These results point out a link between pineal activity and benzodiazepine receptor function in rats. They also indicate that pharmacological doses of melatonin affect benzodiazepine binding sites in rat cerebral cortex.     

[3H]Bicuculline Methochloride Binding to Low-Affinity γ-Aminobutyric Acid Receptor Sites

Abstract: The binding of [³H]bicuculline methochloride (BMC) to mammalian brain membranes was characterized and compared with that of [³H]γ-aminobutyric acid ([³H]GABA). The radiolabeled GABA receptor antagonist showed significant displaceable binding in Tris-citrate buffer that was improved by high concentrations of chloride, iodide, or thiocyanate, reaching >50% displacement in the presence of 0.1 M SCN^?. An apparent single class of binding sites for [³H]BMC (K_D= 30 nM) was observed in 0.1 M SCN^? for fresh or frozen rat cortex or several regions of frozen and thawed bovine brain. The B_max was about 2 pmol bound/mg of crude mitochondrial plus microsomal membranes from unfrozen washed and osmotically shocked rat cortex, similar to that for [³H]GABA. Frozen membranes, however, showed decreased levels of [³H]BMC binding with no decrease or an actual increase in [³H]GABA binding sites. [³H]BMC binding was inhibited by GABA receptor specific ligands, but showed a higher affinity for antagonists and lower affinity for agonists than did [³H]GABA binding. Kinetics experiments with [³H]GABA binding revealed that low- and high-affinity sites showed a similar pharmacological specificity for a series of GABA receptor ligands, but that whereas all agonists had a higher affinity for slowly dissociating high-affinity [³H]GABA sites, bicuculline had a higher affinity for rapidly dissociating low-affinity [³H]GABA sites. This reverse potency between agonists and antagonists during assay of radioactive antagonists or agonists supports the existence of agonist- and antagonist-preferring conformational states or subpopulations of GABA receptors. The differential affinities, as well as opposite effects on agonist and antagonist binding by anions, membrane freezing, and other treatments, suggest that [³H]BMC may relatively selectively label low-affinity GABA receptor agonist sites. This study, using a new commercially available preparation of [³H]bicuculline methochloride, confirms the report of bicuculline methiodide binding by Mohler and Okada (1978), and suggests that this radioactive GABA antagonist will be a valuable probe in analyzing various aspects of GABA receptors.     

Alterations in central neurotransmitter receptor binding sites following estradiol implantation in female rats

The subcutaneous implantation of an estradiol pellet (10 mg) into female rats induced a hypophyseal hyperplasia with hyperprolactinaemia. Examination of neurotransmitter receptors in the hippocampus, striatum and cerebral cortex one month after the implantation revealed that estrogenization was associated with: an increased density of ³H-domperidone binding sites (D₂ receptors) in the striatum and reduced numbers of ³H-serotonin high affinity sites (5-HT₁ receptors) in the hippocampus and of ³H-muscimol binding sites (GABA receptors) in the hippocampus, striatum and cerebral cortex. In contrast, the characteristics of ³H-spiperone binding to 5-HT₂ receptors (in the cerebral cortex) and those of ³H-flunitrazepam binding to benzodiazepine sites (in the three brain regions examined) were not significantly different in estrogenized and in control female rats. However, the enhancing effect of GABA on ³H-flunitrazepam binding was markedly reduced in brain membranes from estrogenized animals. The respective roles of estradiol and prolactin in mediating these changes in neurotransmitter receptors are discussed notably with regard to the regional heterogeneity of estradiol binding capacity in the rat brain.     

Leydig cell receptors for luteinizing hormone releasing hormone and its agonists and their modulation by administration or deprivation of the releasing hormone

Leydig cells isolated from adult rat testes bound ¹²⁵I-labelled luteinizing hormone releasing hormone (LHRH) agonist with high affinity (K_A=1.2 × 10⁹M) and specificity. LHRH and the 3–9 and 4–9 fragments of LHRH agonist competed for binding sites with ¹²⁵I-LHRH agonist but with reduced affinities, whereas fragments of LHRH, and oxytocin and TRH were largely inactive. Somatostatin inhibited binding at high (10^?4M) concentrations but was inactive at 10^?6M and less. Pretreatment of rats for 7 days with 5 μg/day of LHRH agonist reduced binding of ¹²⁵I-LHRH agonist to Leydig cells $\text{in vitro}$ by 25%, whilst inhibition of endogenous LHRH by antibodies for 7 days caused a 40% decrease.     

Endogenous inhibitors of Na+-independent [3H]GABA binding to crude synaptic membranes

The characteristics of the Na⁺-independent high-affinity binding of [³H]GABA to various types of crude synaptic membranes (CSM) prepared from rat brain cortex were studied. In freshly prepared CSM the content of GABA was so high that the high-affinity [³H]GABA binding could not be determined. In contrast when the frozen-thawed CSM were incubated at 37° for 30 min with or without Triton X-100 or phospholipase C and then washed repeatedly, there was a virtual disappearance of GABA from the supernatant extracts and the binding constants of [³H]GABA to CSM could be determined. Two apparent populations of [³H]GABA binding sites, one with a low- and the other with a high-affinity constant, were detected. The ratio of the number of high- to low-affinity binding sites varies with the method used to prepare the membranes. The lowest value of this ratio was observed with membranes incubated at 37° for 30 min. However, when frozen-thawed CSM were treated with 0.05% Triton X-100 repeatedly, the ratio of the number of high- to low-affinity binding sites increased progressively. This increase in ratio is due to a selective increase in the number of the high-affinity sites without significant changes in the number of the low-affinity sites. The extent of the increase in the number of sites that bind [³H]GABA with high affinity after repeated Triton X-100 treatments was paralleled by a decrease of an endogenous protein which inhibits GABA binding. The reapplication of this endogenous material to membranes repeatedly treated with Triton X-100 reduces the number of high-affinity binding sites for [³H]GABA to values similar to those measured in membranes that were not treated with Triton X-100. The inhibitory preparation extracted from CSM incubated with Triton X-100 was shown to be free of GABA or phospholipids. The gel filtration chromatography reveals the presence of two molecular forms of the inhibitor; of these, the high-molecular-weight material fails to bind GABA, whereas the low-molecular-weight material appears to bind GABA. The high-molecular-weight endogenous inhibitor has been termed GABA modulin.     

Studies on the enzymic degradation of luteinizing hormone releasing hormone by rat pituitary plasma membranes

Degradation of luteinizing hormone releasing hormone (LH-RH) by purified plasma membranes from rat pituitaries was investigated. Synthetic LH-RH (0.5 mg/ml) was incubated (20 min, 37°C) with pituitary plasma membranes (750 μg protein/ml). The reaction was stopped by centrifugation at 4°C. The degradation products were isolated by high pressure liquid chromatography using a reversed-phase column. Amino acid analysis of the degradation products indicated that the N-terminal tripeptide (pGlu-His-Trp) and the N-terminal hexapeptide (pGlu-His-Trp-Ser-Tyr-Gly) sequence of LH-RH are the main degradation products. These results suggest that the main cleavage sites of LH-RH by the pituitary plasma membrane-bound enzymes are the Gly⁶-Leu⁷ and the Trp³-Ser⁴ bonds of the neurohormone.     

Isolation, identification and synthesis of an endogenous arachidonic amide that inhibits calcium channel antagonist 1,4-dihydropyridine binding

This study was part of a broad search for endogenous regulators of L-type calcium channels. The screening for active fractions was done by measuring inhibition [³H]1,4-dihydropyridine (DHP) binding to rat cardiac and cortex membranes. An inhibitory fraction, termed lyophilized brain hexane-extractable inhibitor (LBHI), was isolated from hexane extracts of lyophilized calf brain. The active substance was purified by a series of chromatographic steps. ¹³C nuclear magnetic resonance (NMR), ¹H coherence spectroscopy (COSY) NMR and fast atom bombardment (FAB) mass spectroscopy suggested that LBHI was N-arachidonic acid-2-hydroxyethylamide. Synthesis of this substance and subsequent high performance liquid chromatography (HPLC) and NMR analysis confirmed this structure. Synthetic LBHI (SLBHI) inhibited [³H]DHP binding to rat cortex membranes with an IC₅₀ value of 15 μM and a Hill coefficient of 2. Saturation analysis in the presence of SLBHI showed a change in K_D (equilibrium dissociation constant), but not maximal binding capacity (B_max). SLBHI produced an increased dissociation rate, which, along with the Hill slope of > 1, suggested a non-competitive interacton with the DHP binding site. The results suggest that arachidonic acid derivatives may be endogenous modifiers of the DHP calcium antagonist binding site.