Guinea Pig Brain Membranes Research Articles

P-35 - Binding of [3H]NE-100 to sigma ligand binding sites in guinea-pig brain membranes.

P-35 - Binding of [3H]NE-100 to sigma ligand binding sites in guinea-pig brain membranes.

Synthesis and structure-activity studies of N,N'-diarylguanidine derivatives. N-(1-naphthyl)-N'-(3-ethylphenyl)-N'-methylguanidine: a new, selective noncompetitive NMDA receptor antagonist.

Diarylguanidines, acting as NMDA receptor ion channel site ligands, represent a new class of potential neuroprotective drugs. Several diarylguanidines structurally related to N,N'-di-o-tolylguanidine (DTG), a known selective sigma receptor ligand, were synthesized and evaluated in in vitro radioligand displacement assays, with rat or guinea pig brain membrane homogenates, using the NMDA receptor ion channel site specific radioligand [3H]-(+)-5(S)-methyl-10(R),11-dihydro-5H-dibenzo[a,d]cyclohepten-5 ,10- imine (MK-801, 3), and the sigma receptor-specific radioligand [3H]-di-o-tolylguanidine (DTG, 5). This paper presents the structure-activity relationships leading to novel tri- and tetrasubstituted guanidines, which exhibit high selectivity for NMDA receptor ion channel sites and weak or negligible affinity for sigma receptors. The in vitro binding results from symmetrically substituted diphenylguanidines indicated that compounds having ortho or meta substituents (with respect to the position of the guanidine nitrogen) on the phenyl rings showed greater affinity for the NMDA receptor ion channel site compared with para-substituted derivatives. Among the group of ring substituents studied for symmetrical diarylguanidines, an isopropyl group was preferred at the ortho position and an ethyl group was preferred at the meta position. Several unsymmetrical guanidines containing a naphthalene ring on one nitrogen atom and an ortho- or a meta-substituted phenyl ring on the second nitrogen atom, e.g., N-1-naphthyl-N'-(3-ethylphenyl)guanidine (36), showed a 3-5-fold increase in affinity for the NMDA receptor ion channel site and no change in sigma receptor affinity compared to the respective symmetrical counterparts. Additional small substituents on the guanidine nitrogen atoms bearing the aryl rings resulted in tri- and tetrasubstituted guanidine derivatives which retained affinity for NMDA receptor ion channel sites but exhibited a significant reduction in their affinities for sigma receptors. For example, N-1-naphthyl-N'-(3-ethylphenyl)-N'-methylguanidine (40) showed high affinity for the NMDA receptor ion channel site (IC50 = 36 nM vs [3H]-3) and low affinity for sigma receptors (IC50 = 2540 nM vs [3H]-5). Selectivity for the NMDA receptor ion channel sites over sigma receptors appears to be dependent upon the structure of the additional substituents on the guanidine nitrogen atoms bearing the aryl groups. Methyl and ethyl substituents are most preferred in the tri- and tetrasubstituted diarylguanidines. The trisubstituted guanidine, N-1-naphthyl-N'-(3-ethylphenyl)-N'-methylguanidine (40) and its close analogues showed good in vivo neuroprotection and are potential neuroprotective drug candidates for the treatment of stroke and other neurodegenerative disorders.

Selective labeling of κ2 opioid receptors in rat brain by [ 125I]IOXY: Interaction of opioid peptides and other drugs with multiple κ2a binding sites

Recent studies from our laboratory resolved two subtypes of the κ 2 binding site, termed κ 2a and κ 2b, using guinea pig, rat, and human brain membranes depleted of μ and δ receptors by pretreatment with the site-directed acylating agents BIT (μ-selective) and FIT (δ-selective). 6β-Iodo-3,14-dihydroxy-17-cyclopropylmethyl-4,5α-epoxymorphinan (IOXY), an opioid antagonist that has high affinity for κ 2 sites, was radioiodinated to maximum specific activity (2200 Ci/mmol) and purified by high pressure liquid chromotography and used to characterize multiple κ 2 binding sites. The results indicated that [ 125I]IOXY, like [ 3H]bremazocine, selectively labels κ 2 binding sites in rat brain membranes pretreated with BIT and FIT. Using 100 n M [ d-Ala 2-MePhe 4,Gly-ol 5]enkephalin to block [ 125I]IOXY binding to the κ 2b site, two subtypes of the κ 2a binding site were resolved, both in the absence and presence of 50 μ M 5′-guanylyimidodiphosphate. Viewed collectively, these results provide further evidence for heterogeneity of the κ opioid receptor, which may provide new targets for drug design, synthesis, and therapeutics.

Model for the interaction of crotoxin, a phospholipase A2 neurotoxin, with presynaptic membranes.

Crotoxin is a phospholipase A2 neurotoxin that impairs the release of acetylcholine at neuromuscular junctions, primarily at the presynaptic level. It associates a phospholipase A2 subunit, CB, with a chaperon subunit, CA. We have shown elsewhere that the purely cholinergic synaptosomes from the Torpedo electric organ provided a convenient model to study the pharmacology of crotoxin and other related neurotoxins [Délot, E., & Bon, C. (1992) J. Neurochem. 58, 311-319]. In the present experiments, we labeled crotoxin with 125I and demonstrated saturable binding to Torpedo presynaptic membranes. In the range of concentrations that was effective on synaptosomes, crotoxin bound to a single class of sites without cooperativity. The binding was affected by divalent cations, and its kinetics was rather complex. We observed a competition between crotoxin and related neurotoxins, but not CB. Although CA could not bind, it could compete efficiently with crotoxin. We compare our results with those previously obtained by others on guinea pig brain membranes. On Torpedo membranes, as on all models tested, CB was the major species bound to the membrane, while CA remained in solution. However, the mechanism underlying this phenomenon had never been clarified. The major question is the time scale of the events, i.e., does CB bind before or after dissociating from CA? Our results indicate that the predominant pathway involves the formation of a ternary complex between crotoxin's subunits and the acceptor site preceding the release of CA.

N,N′-disubstituted acetamidines as high affinity sigma receptor ligands

A series of N,N′-disubstituted acetamidines was prepared as mammalian brain sigma receptor ligands. The in vitro binding affinities of seven representative ligands were measured using guinea pig brain membrane suspensions. N-1-adamantyl- N′- o-tolyl acetamidine is the most potent ligand in the series (IC 50 = 6 nM vs [ 3H]=DTG). All ligands showed low affinity toward the PCP site.

Haloperidol treatment differentially regulates [ 3H]DTG and [ 3H](+)-3-PPP labeled σ binding sites

The effect of repeated haloperidol administration on σ binding sites in brain membranes was assessed using [ 3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ((+)-3-PPP) and [ 3H]1,3-di-o-tolylguanidine (DTG). Administration of haloperidol (1 mg/kg, i.p.) to guinea pigs for 14 consecutive days followed by a 4 day drug-free period prior to sacrifice resulted in 75% and 6% decreases in the specific binding of [ 3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine and [ 3H]1,3-di-o-tolylguanidine, respectively, when measured using a single concentration (2 nM) of radioligand. Scatchard analysis revealed a reduction in both the maximum number of [ 3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine binding sites and the affinity of these sites for the radioligand; the potency of 1,3-di-o-tolylguanidine to inhibit [ 3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine binding was also reduced. In parallel studies, the potency of 1,3-di-o-tolylguanidine to inhibit [ 3H]1,3-di-o-tolylguanidine binding was unaffected by haloperidol treatment, but the potency of (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine against [ 3H]1,3-di-o-tolylguanidine was reduced 3-fold. Phenytoin, which increased (10-fold) the potency of dextromethorphan to inhibit [ 3H]1,3-di-o-tolylguanidine binding in control membranes, had no effect in membranes obtained from haloperidol-treated animals. The reduction in [ 3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine binding was dependent upon the duration of the drug-free period and amounted to 73% and 25% in brain membranes prepared from animals sacrificed 14 days and 28 days, respectively, following cessation of drug treatment. Repeated administration of other antipsychotic and σ agents including DTG, dextromethorphan, spiperone, chlorpromazine and clozapine had no effect on [ 3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine or [ 3H]1,3-di-o-tolylguanidine binding. These findings suggest that repeated haloperidol administration selectively regulates [ 3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine binding in guinea pig brain membranes. However, the observations that repeated drug treatment resulted in (1) reduced affinity of (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine for [ 3H]1,3-di-o-tolylguanidine binding sites, and (2) a loss of the ability of phenytoin to increase dextromethorphan's potency against [ 3H]1,3-di-o-tolylguanidine, suggest that [ 3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine and [ 3H]1,3-di-o-tolylguanidine binding sites are distinct but functionally coupled.

Synthesis and evaluation of conformationally restricted N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamines at sigma receptors. 2. Piperazines, bicyclic amines, bridged bicyclic amines, and miscellaneous compounds.

As a continuation of our earlier study (J. Med. Chem. 1992, 35, 4334-4343) we conformationally restricted the sigma-receptor ligand 2-(1-pyrrolidinyl)-N-[2-(3,4-dichlorophenyl)ethyl]-N-methylethylamine (1) by incorporating it into a series of homologous piperazines 3-9 and homopiperazines 10 and 11, diazabicyclononanes and decanes, bridgehead bicyclooctanes and nonanes as well as other miscellaneous compounds. sigma-Receptor binding affinities were obtained using [3H](+)-pentazocine in guinea pig brain membrane sigma 1 sites. The studies suggest that the nitrogen lone pair orientation found in the piperazines affords the strongest binding interaction. Other nitrogen lone pair orientations or compounds representing unlikely staggered conformations of 1 [ as in 4-[2-(3,4-dichlorophenyl)ethyl]-1,4-diazabicyclo[3.2.2]nonane (16)] show very weak sigma interaction. Comparison of the binding data of different N-substituted homologues of 1 with those of the 1-[2-(3,4-dichlorophenyl)ethyl]-4-alkylpiperazines suggests that the two nitrogen atoms of 1 are working in opposition to one another in terms of their sensitivity to steric bulk. The high binding affinity of the 1,4-diazabicyclo[4.3.0]nonanes 12 suggests that these may approximate the methyl and pyrrolidine ring conformations found in 1 when it is bound to the sigma receptor. Compound 12 exhibited a 4-fold enantioselectivity favoring (+)-12. The synthesis of 6,7-dichloro-2-[[2-(1-pyrrolidinyl)ethyl]amino]tetralin (19) and its desmethyl derivative 20 permitted constraint of the 3,4-dichlorophenyl and N-methyl moieties of 1 into a gauche orientation. The binding data suggests that this conformation in 1 favors strong binding interaction at sigma-receptors. sigma-Receptor Ki's ranged from 0.55 nM for 1-[2-(3,4-dichlorophenyl)ethyl]-4-n-butylpiperazine (7) to 654 nM for 16. Overall comparison of the results indicate that 1 is subject to considerable conformational freedom and suggests that the sigma receptor is not subject to rigid stereochemical restraints with 1. These results add to our earlier study where we restrained 1 using simple monocyclic heterocycles.

Binding of [ 3H]FH-510 to σ ligand recognition sites in guinea-pig brain membranes

We examined the characteristics of the binding of radiolabeled 5,8-dimethyl-4-(2-di-n-propylaminoethyl)carbazol monohydrochloride ([ 3H]FH-510), a highly potent and selective σ ligand, to guinea-pig brain membranes. [ 3H]FH-510 showed saturable and reversible binding to σ binding sites. The association rate constant (k +1) and dissociation rate constant (k −1) and dissociation (k −1) of [ 3H]FH-510 were 0.23 min −1·nM −1 and 0.081 min −1, respectively. Scatchard plot analysis showed a dissociation constant (K d and maximal number of binding sites (B max) of 6.0 ± 0.63 nM and 1763.3 ± 177.4 fmol/mg protein (n = 7), respectively. The rank order of potency (K i) of several structurally dissimilar σ ligands obtained for the displacement of [ 3H]FH-510 binding was highly correlated with that determined for [ 3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ([ 3H](+)-3-PPP) binding. The binding of [ 3H]FH-510 was not influenced by histaminergic, dopaminergic, adrenergic, serotonergic or cholinergic agents at 10 −7 M. Higher [ 3H]FH-510 binding to brain regions was observed in the cerebellum and pons-plus-medulla. Except for the nuclear fraction, the highest level of [ 3H]FH-510 and [ 3H](+)-3-PPP binding to subcellular fractions was observed in the microsomal fraction. From these results, it is suggested that FH-510 selectivity binds with high affinity to σ binding sites in guinea-pig membranes.

On the significance of the C-terminal primary amide in cholecystokinin

We report on the synthesis of some analogs of the C-terminal heptapeptide of cholecystokinin in which the C-terminal primary amide has been deleted and the L-tryptophan replaced by a D-tryptophan. These analogs were evaluated in vitro for their ability (i) to recognize the CCK receptor on rat pancreatic acini and on guinea pig brain membranes; (ii) to stimulate amylase release from rat pancreatic acini. Although previous works suggested that the C-terminal primary amide is essential for exhibiting a full biological response on pancreatic acini, this study does not confirm this hypothesis.

Comparison of κ2-opioid receptors in guinea pig brain and guinea pig ileum membranes

The presence of κ-opioid receptor subtypes has been clearly established in guinea pig brain. Using [ 3H]bremazocine in the presence of reversible blockers of μ, δ and κ 1 receptors, two additional binding sites can be determined in guinea pig brain membranes. The site with higher affinity for the opioid ligands represents κ 2, while the other site has low affinity and is poorly characterized. The κ 2 site has high affinity for ethylketocyclazocine and other benzomorphans, as well as for the dynorphin gene products tested. The dynorphin analogs have no appreciable affinity for the low affinity site, so this site should not be called a κ receptor. With an appropriate membrane preparation, κ 2 binding can also be demonstrated in the guinea pig ileum. Binding affinities for selected ligands at κ 2 in guinea pig ileum membranes are very similar to affinities found in brain membranes.

Relationship between kappa 1 opioid receptor binding and inhibition of adenylyl cyclase in guinea pig brain membranes

Previously, we showed that k- selective ligands inhibit adenylyl cyclase in guinea pig cerebellar membranes. The present studies explore the relationship between k 1 binding sites (as determined with [ 3H]U-69,593 binding) and k 1- inhibition of adenylyl cyclase (using U-50,488H) in guinea pig brain membranes. Various k opioids displaced [ 3H]U-69,593 binding at a single site with subnanomolar affinities. These agonists were several hundred-fold weaker in inhibiting adenylyl cyclase, but for most agonists the rank order of adenylyl cyclase inhibition paralleled the displacement of k 1 binding. The correlation of ic 50 values for both adenylyl cyclase and binding was significant except for α-neo endorphin, which was relatively weak at inhibiting adenylyl cyclase despite a K i value of 0.08 nM versus k 1 binding. Comparison between k 1 binding and k 1- inhibited adenylyl cyclase across eleven guinea pig brain regions revealed that k 1- inhibited adenylyl cyclase was highest in the cerebellum, absent in thalamus and superior colliculus, and moderate in other regions. In most regions, k 1 binding correlated with the efficacy of k 1- inhibited adenylyl cyclase. However, the hippocampus had high levels of k 1 -inhibited adenylyl cyclase despite low levels of k 1 binding, while cortex exhibited a high density of k 1 sites but a relatively low level of k 1- inhibited adenylyl cyclase. Reaction of cerebellar k receptors with β-chlornmaltrexamine (β-CNA) blocked both k 1 binding and k 1- inhibited adenylyl cyclase. The effect of β-CNA on k 1- inhibited adenylyl cyclase was to inhibit efficacy with little decrease in agonist potency, thus suggesting no significant level of k receptor reserve for this effector system.

P-380 - Binding of [3H]FH-510 to sigma ligand binding sites in guinea pig brain membranes.

P-380 - Binding of [3H]FH-510 to sigma ligand binding sites in guinea pig brain membranes.

Characterization of the binding of [ 3H](+)-pentazocine to σ recognition sites in guinea pig brain

The selective σ compound (+)-pentazocine was radiolabeled and its binding characteristics in guinea pig brain membranes were investigated. [ 3H](+)-Pentazocine bound to a single high-affinity site with a K D of 2.9 nM and a B max of 1998 fmol/mg protein. Saturation was achieved at a ligand concentration of 15 nM. Maximal specific binding was observed at 37°C and was greater than 90% of total binding. Equilibrium was reached by 120 min and dissociation was complete by 420 min, with a t 1,2 of 121 min. Li +, Ca 2+ and Mg 2+ inhibited binding at high concentrations, and binding was insensitive to adenyl and guanyl nucleotides. Stereoselectivity was observed for the inhibition of binding by benzomorphans, 3-(3-hydroxyphenyl)-N-propylpiperidine and butaclamol, and the (+) enantiomers and α diastereomers of pentazocine and cyclazocine were more potent than their corresponding (−) enantiomers and β diastereomers. The rank order of potency for the σ reference agents to displace [ 3H](+)-pentazocine binding was similar to that reported using the [ 3H]σ ligands dextromethorphan, 1,3-di(2-tolyl)guuanidine and (+)-3-(3-hydroxyphenyl)-N-propylpiperidine. Haloperidol, (+)-pentazocine, (+)-3-(3-hydroxyphenyl)-N-propylpiperidine and rimcazole were competitive inhibitors of binding to the [ 3H](+)-pentazocine-defined σ recognition site, suggesting that these different structural classes of compounds all bind to a single molecular entity.

Characterization of NK-1 receptors in guinea pig and rat brain membranes with NK-1 peptides and a non-peptide antagonist

The major finding of the present investigation is the demonstration of different NK-1 receptors in rat and guinea pig brain membranes with CP 96345 (non-peptide NK-1 antagonist) and R-544 (NK-1 peptide antagonist). We used [ 3H]Sar 9, Me(O 2) 11]SP, the highly selective ligand for NK-1 receptor to compare NK-1 binding sites in rat and guinea pig brain membranes. Scarchard analysis revealed the existence of a single population of [ 3H][Sar 9, Met(O 2) 11]SP binding sites in both preparations. The affinity and the maximal number of bindind sites were found closely similar in rat ( K d 2 nM, B max = 37 fmol/mg protein) and guinea pig brain membranes ( K d = 3 nM, B max = 25 fmol/mg of protein). The order of potency of neurokinins to inhibit [ 3H][Sar 9, Met(O 2) 11]SP binding from rat brain (SP > NKA > NKB) was found different of that observed on guinea pig brain (SP > NKB > NKA). Results obtained with [Sar 9, Met(O 2) 11]SP, [βAla 8]NKA(4–10) and [MePhe 7]NKB suggest that selective agonists cannot discriminate between NK-1 receptors of different species. Using the non-peptide antagonist CP 96345 and the tripeptide R-544, we found that these two NK-1 antagonists discriminate between rat and guinea pig [ 3H][Sar 9, Met(O 2) 11]SP binding sites.

Multiple sigma binding sites in guinea-pig and rat brain membranes: G-protein interactions.

1. Evidence is accumulating for multiple sigma (sigma) sites in the mammalian CNS. 2. We have addressed this problem and have examined sigma site - G-protein coupling in guinea-pig and rat brain membranes. 3. Ditolylorthoguanidine (DTG), (+)-3-(3-hydroxyphenyl)-N-1-(propyl)piperidine (3PPP) and dextromethorphan displaced [3H]-DTG (3.4 nM) with low Hill slopes of 0.5, 0.6 and 0.6, respectively in guinea-pig brain membranes. 4. In the presence of 5'-guanylylimidodiphosphate (Gpp(NH)p; 100 microM), the specific binding of [3H]-DTG was reduced by 36.7%, the Hill slope of 3PPP was increased to near unity, the ability of dextromethorphan to displace DTG was virtually abolished and the Hill slope for DTG remained low (0.7), indicating the presence of at least two binding sites. These data indicate that although Gpp(NH)p removes a dextromethorphan high affinity site, two DTG selective sites remain in the presence of Gpp(NH)p. 5. The present study suggests that DTG binds to at least three sites in guinea-pig brain membranes, at least one of which is G-protein linked. 6. In rat brain membranes, DTG displaced itself (3.4 nM) with a Hill slope near 1. 3PPP displacement of [3H]-DTG was comparable with the guinea-pig (Hill slope 0.5) and displaced from more than 1 site. Dextromethorphan did not displace [3H]-DTG at concentrations below 10 microM. 7. The heterogeneity of sigma sites appears to be less in rat than in guinea-pig brain membranes.

Opioid agonist and antagonist activities of morphindoles related to naltrindole.

A series of naltrindole-related ligands (4-10) with an N-methyl,N-phenethyl,N-cinnamyl, or an unsubstituted basic nitrogen were synthesized and tested for opioid agonist and antagonist activity in smooth muscle preparations and in mice. The nor compounds (4 and 6) and the phenethyl derivatives (5 and 8) displayed full agonist activity (IC50 = 85-179 nM) in the mouse vas deferens preparation (MVD) while the other members of the series exhibited partial agonist or weak antagonist activity. In the guinea pig ileum preparation (GPI), all compounds except 8 were partial agonists. The ligands that were evaluated in mice were found to produce antinociception that was not selectively mediated via delta opioid receptors. However, two of these ligands (4 and 5) appeared to be delta-selective opioid receptor antagonists at subthreshold doses for antinociception. The finding that all of the compounds bind selectively to delta opioid receptors in guinea pig brain membranes together with the in vitro pharmacology and in vivo antagonist studies suggests that the lack of delta agonist selectivity in vivo may be due to a number of factors, including a basic difference between the delta receptor system in the MVD and in the mouse brain. Further, it is suggested that the constellation of message and address components in the morphindole nucleus may tend to stabilize delta receptors in the brain in antagonist state.

A pharmacological profile of glycopyrrolate: Interactions at the muscarinic acetylcholine receptor

1. 1. Glycopyrrolate, a synthetic quaternary ammonium compound had biphasic effects on the contraction of guinea-pig atrium. At concentrations between 0.4 and 20 μM, glycopyrrolate induced a small but consistent increase in the contraction force. 2. 2. Further increase in the concentration of glycopyrrolate produced a concentration-dependent reduction in the force of contraction with a EC 50 of 0.24 mM. 3. 3. This negative inotropic effect was opposed by a Ca 2+ channel agonist, Bay K 8644. Glycopyrrolate also antagonized potently the depressant effects of carbachol and acetylcholine in guinea-pig atrium. 4. 4. Schild analysis showed a pA 2 value of 8.16 against carbachol and 8.39 against acetylcholine. These similar pA 2 values suggested that both compounds may have a common interacting site. The interactions however cannot be explained by a simple competition model as the slopes of the Schild plots were larger than unity. 5. 5. The mutual competition between glycopyrrolate and quinuclidinyl benzilate (QNB), gallamine or methoctramine indicated that glycopyrrolate could have multiple action sites in the atrium. Interaction at an allosteric site was implicated. 6. 6. Radioligand binding studies showed that glycopyrrolate displaced pirenzepine (PZ) and AF-DX 384 from their binding to the M 1 and M 2 muscarinic receptors in guinea-pig brain membranes respectively. 7. 7. The respective binding constants ( K is) were 0.60 and 0.03 nM. The Hill coefficient value ( n H) for glycopyrrolate against [ 3H] PZ was larger than unity, suggesting positive cooperativity at the receptor complex. In contrast, the n H of the agonist [ 3H] AF-DX 384 was not different from unity, indicating a simple competitive inhibition. 8. 8. Our results confirm the antimuscarinic properties of glycopyrrolate. We suggest that the therapeutic value of glycopyrrolate in the treatment of organophosphate poisoning is associated with its potency against prolonged muscarinic receptor activation by cholinergic agonists.

Delta opioid antagonist activity and binding studies of regioisomeric isothiocyanate derivatives of naltrindole: evidence for delta receptor subtypes.

The isothiocyanate group was attached to the 4'-, 5'-, 6'-, or 7'-position of naltrindole in an effort to determine the importance of the position of this electrophilic group on the selectivity for subtypes of delta opioid receptors. All of the ligands were delta-selective when tested against standard agonists in smooth muscle preparations. However, the rank-order delta antagonism of antinociception in mice did not parallel the in vitro pharmacologic data. The 5'-isothiocyanate 2 was the most potent and selective antagonist in vivo, causing a 52-fold increase of the ED50 for [D-Ser2,D-Leu5]enkephalin-Thr6 (DSLET) and no increase for [D-Pen2,D-Pen5]enkephalin (DPDPE). The effect of each of the ligands on the binding of [3H]DSLET and [3H]DPDPE to guinea pig brain membranes clearly differentiated between the binding sites that recognize these radioligands. These studies provide additional evidence for the presence of two subtypes of delta opioid receptors.

New prenylamine analogues: synthesis and Ca 2+-entry blocking activity

The synthesis of a series of diphenylalkylamine derivatives related to prenylamine is reported. The amphetamine group in the prenylamine structure was replaced by other moieties. In addition to substitutions in the aromatic rings, heteroatoms such as sulphur and oxygen were introduced in the chain. The calcium-entry blocking activity was assayed in binding experiments on a guinea-pig brain membrane preparation by displacing [ 3H]-nitrendipine.

A simple, highly sensitive assay for measurement of digitonin during receptor solubilization

A simple and highly sensitive assay for measuring total digitonin in biological samples is described. The assay is based on the ability of digitonin to hemolyze red blood cells. The precision and reproducibility of the assay was excellent with intra- and inter-assay variabilities of < 1% and 6%, respectively. The assay was used to evaluate several potential methods for removing digitonin from biological samples (digitonin extracts from guinea pig brain membranes). Dialysis and G-25 Sephadex chromatography were ineffective. However, protein and digitonin can be effectively separated by ammonium sulfate precipitation followed by dialysis. The κ I opioid receptor survived these procedures with no change in affinity for [ 3H]U-69,593. In conclusion, the hemolytic assay for digitonin appears to provide a practical means for determining detergent concentrations during receptor purification and characterization and for evaluating potential methods for detergent removal. Although an in depth analysis of the assay was carried out only for digitonin, CHAPS and deoxycholate also caused 50% hemolysis at concentrations well below those commonly used for receptor solubilization and, therefore, the general assay procedures might have applicability for measurement of these and perhaps other detergents used in receptor solubilization as well.