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A series of 2-phenylimidazo[1,2-b]pyridazine derivatives were synthesized and evaluated for their in vitro anthelmintic activity against Haemonchus contortus. The most active compounds had in vitro LD99 values of 30 nM, which is comparable to that of the benchmark commercial nematocide, Ivermectin.

A series of novel 2-alkoxy- and 2-aryloxyiminoalkyl trifluoromethanesulfonanilide derivatives have shown significant in vitro parasiticidal activity against the ectoparasites Ctenocephalides felis and Rhipicephalus sanguineus. A number of these compounds also displayed significant in vitro endoparasite activity against the nematode Haemonchus contortus.

A series of 2-phenyl-3-(1H-pyrrol-2-yl)acrylonitrile derivatives were synthesized and evaluated for in vitro activity against the endoparasite Haemonchus contortus and the ectoparasite Ctenocephalides felis. Some compounds had significant in vitro activity against these parasites.

The binding characteristics of the sigma ligand [3H]1.3-di(2-tolyl)guanidine (DTG) were investigated in membranes prepared from the Jurkat T cell line. Binding was saturable with a KD of 56 +/- 3 nM and a Bmax of 11706 +/- 3173 fmol/mg protein (n = 3). The rank order of potency for sigma reference compounds to inhibit binding in the Jurkat cell line was ifenprodil > 1,3-di(2-tolyl)guanidine > haloperidol > carbetapentane > (+)3-(3-hydroxyphenyl)-N-propylpiperidine ((+)3-PPP) > (-)pentazocine > caramiphen > (+)pentazocine, and significantly correlated with potency at sigma 2 binding sites in guinea pig brain (r = 0.90, p < 0.01). The immunomodulatory activities of the sigma ligands 1,3-di(2-tolyl)guanidine, haloperidol. (-)pentazocine and (+)pentazocine on CD3-induced proliferation, IL-2 production and Ca2+ flux in human lymphocytes did not reveal any consistent pharmacology that could be ascribed to potency of these compounds at sigma binding sites. Collectively the data demonstrate that the [3H]1,3-di(2-tolyl)guanidine binding site on Jurkat cell membranes has a pharmacology consistent with sigma receptors, but no modulation of functional activity or intracellular events in human peripheral blood lymphocytes correlating with sigma receptors was found.

The preparation of a series of quinazoline-2,4-diones, 1-3, and pyrrolo[3,4-d]pyrimidine-2,4-diones, 4-8 is described. A small number of quinazolinedione analogs were identified from random screening to possess low micromolar (1.3-4.4 microM) potency in the nuclear factor of activated T cells-1-regulated beta-galactosidase expression assay. An expanded analog search resulted in identifying pyrrolopyrimidinedione 4b which is 5-10-fold (0.26 microM) more potent than the quinazolinediones. Replacement of the benzyl group with naphthyl led to greater potency and conformationally restricted analogs 4u-w. The naphthyl and acenaphthyl analogs are 10-100 times more potent inhibitors of beta-galactosidase expression than 4b. Binding affinity data for displacement of radiolabeled 4s from Jurkat cell membranes reflected an excellent correlation with the IC50 value for inhibition of beta-galactosidase activity. These products, whose structure-activity relationships are discussed, are of interest as potential agents for preventing interleukin-2 gene transcription.

A series of competitive, nonpeptide bradykinin receptor antagonists based on an α-amino acid scaffold have been developed and biologically characterized. The lead compound in the series, WIN 64338, demonstrates competitive inhibition of bradykinin-mediated functional responses through B2 receptors in a variety of tissues and species. WIN 64338 is specific for the bradykinin B2 receptor; it is inactive at both the B1and B3 kinin receptors. In conscious guinea pigs, WIN 64338 inhibits kinin-mediated bronchoconstriction but does not attenuate a similar response to acetylcholine. A series of WIN 64338 analogues display a well-defined structure–activity relationship, strongly suggesting binding in a specific manner to the B2 receptor. Structure–activity data suggest that a hydrophobic binding pocket that prefers large aromatic groups in a specific conformational orientation exists in the receptor ligand binding domain. This class of nonpeptide bradykinin receptor antagonists may lead to the design of other compounds with enhanced receptor affinity and optimal in vivo biological activity.Key words: bradykinin, antagonists, nonpeptide.

The protein tyrosine kinase p56Ick, which is expressed predominantly in lymphocytes, plays a critical role in optimal T cell activation through the T cell antigen receptor. An approach is presented for the discovery of selective p56Ick inhibitors, which are potential immunosuppressants. A non-radioactive assay for p56Ick tyrosine kinase activity has been developed and adapted for high volume screening. This assay does not require purified enzyme. p56Ick in the plasma membranes of a human T cell line is purified in situ by immobilization onto the wells of a microtiter plate using an antibody specific for p56Ick. Following the kinase reaction in the presence of test compound, autophosphorylated p56Ick is detected with a biotinylated monoclonal antibody to phosphotyrosine. Using the approach described in this report, three simple chromones have been identified that inhibit p56Ick autophosphorylation with low micromolar potencies and exhibit some selectivity for p56Ick over the serine/threonine and other tyrosine kinases tested. These compounds constitute a novel group of p56Ick tyrosine kinase inhibitors. ©1995 Wiley-Liss, Inc.

Angiotensin converting enzyme (ACE) degrades both angiotensin II and bradykinin. Accordingly, we hypothesize that ACE inhibitors can serve as models to design antagonists for the bradykinin receptor. The potent ACE inhibitor Quinapril was modified to serve as a spacer separating two lipophilic positive charges required for bradykinin binding. The synthesis and bradykinin receptor activity of a series of antagonists 2–10 based on this hypothesis is described in this report.

A new p56idk tyrosine kinase inhibitor WIN 61651 [1,4-dihydro-7-(4-methyl-1-piperizinyl)-1-(4-(4-methyl-1-piperizinyl))phenyl-4-oxo-3-quinolinecarboxamide) is described. WIN 61651, which is competitive with AP, demonstrates selectivity for the lymphoid restricted tyrosine kinase p56lck over serine/threonine kinases, such as protein kinase C and protein kinase A, and over some other tyrosine kinases, including erbB2, epidermal growth factor receptor, and insulin receptor; however, it is equipotent for inhibition of p56lck and the platelet derived growth factor receptor tyrosine kinases. WIN 61651 inhibits p56lck activity in cell-free assays, tyrosine kinase activity in a T lymphocytic cell line, and T cell activation, as measured by IL-2 production by purified CD4 positive peripheral blood T lymphocytes and the mixed lymphocyte reaction. WIN 61651 constitutes a new tool for studies on the role for tyrosine kinases in lymphocyte function.

AMPA receptors may play an important role in acute and chronic neurodegenerative diseases. An assay for the specific binding of [3H]-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) to receptors in membranes from post-mortem human brain is described, which can be used in screening for selective AMPA receptor antagonists. Membranes were prepared from frozen human adult hippocampus and whole fetal brain tissues. [3H]-AMPA binding to human hippocampus was saturable; Scatchard analysis of equilibrium binding data indicated high and low affinity sites with affinity binding constants (KD) of 3.4 +/- 0.5 nM and 65 +/- 9 nM (n = 7) respectively. Biphasic association and dissociation rate constants for [3H]-AMPA binding were consistent with the biphasic Scatchard analysis. Inhibition of [3H]-AMPA binding revealed a rank order of potency as quisqualate = AMPABOAAL-glutamate = DNQX = CNQXkainateL-aspartate = NMDA. AMPA receptors in human fetal brain had a comparable pharmacology. AMPA/kainate receptors were expressed in frog oocytes following injection of RNA prepared from human fetal brain. Human brain tissues may therefore be utilized for screening and functional analysis of AMPA receptor antagonists.

Bradykinin, desArg 9 BK, some agonist analogues and several antagonists have been tested in isolated organs in order to identify bradykinin B 2 receptor subtypes. The initial pharmacological characterization was made in the rabbit jugular vein and the guinea pig ileum, two widely used B 2 preparations which have shown marked differences in their sensitivities to both agonists and antagonists. The study has then been extended to peripheral tissues (stomach, colon, urinary bladder) of four species (the rat, guinea pig, rabbit and man) and to isolated vessels (rabbit jugular vein, rabbit vena cava, guinea pig pulmonary artery, rat portal vein) in order to determine if pharmacologic receptor subtypes may be related to species. It has been shown that B 2 receptors in rat and guinea pig tissues belong to a similar pharmacological entity, a receptor which is different from that mediating the responses of rabbit and human tissues. Agonists order of potency ([Hyp 3 ]BK > BK > [Aib 7 ]BK) obtained in the rabbit jugular vein is different from that found in the guinea pig ileum (BK ≤ [Aib 7 ]BK > [Hyp 3 ]BK). Affinities of competitive antagonists (for instance DArg[Hyp 3 ,DPhe 7 ,Leu 8 ]BK) in rabbit tissues are higher than in guinea pig and rat tissues by at least 2 log units, while the non peptidic compound WIN 64338 is more active (also by two log units) in guinea pig than in human and rabbit tissues. The non competitive long-acting antagonist HOE 140 is very potent and equally active in the four species. Some antagonists (peptides without unnatural residues, peptides with unnatural residues, non peptides) have been shown to be specific for kinin receptors and selective for the B 2 . Altogether, the present results a) confirm the existence of two B 2 receptor subtypes, b) suggest that receptor subtypes may be species dependent and c) indicate that the B 2 receptor subtype found in the rabbit is similar to that found in man.

A variety of the title imidazopyridazine derivatives are synthesized and evaluated for their in vitro anthelmintic activity.

[3H]Bradykinin binds to intact human IMR-90 fetal lung fibroblasts in a time and dose-dependent manner. Binding equilibrium was attained by 120 minutes at 4 degrees C. [3H]Bradykinin binding was saturable; Scatchard analysis of saturation binding data demonstrated a single binding site having a KD = 1.8 +/- 0.2 nM and a receptor concentration of 17.4 +/- 4.0 fmol/10(5) cells. The calculated value for KD(k-1/k1) from the association (k1 = 4.71 x 10(6) mol-1 min-1) and dissociation (k-1 = 1.13 x 10(-2) min-1) rate constants was 2.4 nM. The rank order of potency observed for bradykinin peptide agonists, bradykinin > Lys-bradykinin > Met,Lys-bradykinin > Ile,Ser-bradykinin >> des-Arg9-bradykinin, is consistent with that of a bradykinin B2 receptor. Bradykinin stimulated efflux of 45Ca2+ from IMR-90 cells dose dependently with an EC50 = 331 +/- 50 pM. 45Ca2+ efflux was also demonstrated with Lys-bradykinin and Met-Lys-bradykinin but not by des-Arg10-kallidin (100 nM) or NKA (1 microM). Hoe-140 inhibited bradykinin-induced 45Ca2+ efflux (IC50 = 3 +/- 2 nM). D-Phe7-substituted bradykinin analogues stimulated 45Ca2+ efflux dose dependently and this stimulation of 45Ca2+ efflux was inhibited by Hoe-140. These results suggest that D-Phe7 substituted bradykinin analogues are agonists at the bradykinin B2 receptor in IMR-90 cells.

Receptor type and function of bradykinin (BK) receptors on human synovial fibroblasts (HSF) was determined. Scatchard analysis of [3H]BK saturation binding to intact synovial cells revealed a single binding site, with a Kd of 3.8 +/- 0.6 nM. HSF express approximately 50,000 BK sites/cell. Specificity of [3H]BK binding was confirmed by the ability of several BK peptide agonists and antagonists to inhibit binding in a dose dependent manner. The rank order of potency for agonist inhibition of [3H]BK and the inability of selective antagonists of the B1-type to displace binding suggest that the BK receptor on HSF is a B2 subtype receptor. The addition of BK to HSF caused a time and concentration dependent increase in PGE2 production. This BK induced PGE2 production was blocked by specific B2 type BK antagonists and not by B1 antagonists. The results of this study identify B2 type BK receptors on synovial fibroblasts and suggest that BK may be a primary mediator in inflammatory arthritis.

A series of hydrazonotrifluorosulfonanilide derivatives were synthesized and evaluated for in vitro activity against the ectoparasites Ctenocephalides felis and Rhipicephalus sanguineus. Some compounds with excellent activity against tick were identified.

We report the synthesis and molecular characterization of a biotinylated analog of kallidin, [Lys]bradykinin. Bradykinin was prepared by solid phase peptide synthesis. Before cleavage from the resin, a biotin moiety was coupled to the epsilon amino group of a lysine in the zeroth position of the bradykinin peptide. An omega-amino caproic acid spacer was incorporated between the biotin group and the N-terminal lysine. The biotinylated peptide was deprotected, cleaved from the resin and purified by RP-HPLC. The identity of this analog was confirmed by amino acid analysis and FAB-mass spectrometry. Biotinyl [Lys]bradykinin (BLBK, mol, wt. = 1528) inhibited [3H]-bradykinin binding to guinea pig ileum homogenates dose dependently, with an IC50 of 28.9 +/- 6 nM. The IC50 for [Lys]bradykinin was approximately 10-fold lower, 3.2 +/- 0.6 nM. BLBK induced contractility in an isolated guinea pig smooth muscle preparation with an EC50 of 129 +/- 14 nM; the corresponding value for [Lys]bradykinin was 29 +/- 8 nM. These data are consistent with the difference in binding potency observed for BLBK compared to [Lys]bradykinin. In an ELISA assay using BLBK and affinity-purified rabbit anti-bradykinin antibody, BLBK bound to anti-bradykinin antibody with an EC50 = 1.21 +/- 0.54 nM. Rank order potencies for several bradykinin peptide analogs suggest that the epitope on bradykinin recognized by the antibody is likely to be at the carboxy terminus of the peptide.

In this report, we describe the discovery and characterization of a novel biarylhydrazone series of platelet-derived growth factor (PDGF) receptor tyrosine kinase inhibitors typified by the prototype WIN 41662 (3-phenyl-N1-[1-(4-pytidyl)pyrimidine]hydrazone). WIN 41662 inhibited PDGF-stimulated autophosphorylation of PDGF receptors from human vascular smooth muscle cells (hVSMC) with an IC50 value of 60 nM. The inhibitor appeared to be competitive with respect to substrate (Mn(2+)-ATP), having a calculated Ki of 15 +/- 5 nM. WIN 41662 was approximately 500-fold more potent in inhibiting the PDGF receptor tyrosine kinase than the p56lck tyrosine kinase. It was inactive against other serine/threonine and tyrosine kinases tested. WIN 41662 produced concentration-dependent inhibition of PDGF-stimulated receptor autophosphorylation in intact hVSMC with an IC50100 nM. Intracellular Ca2+ mobilization and cell proliferation were events that occurred in hVSMC subsequent to PDGF receptor activation. WIN 41662 inhibited PDGF-stimulated Ca2+ mobilization and cell proliferation ([3H]TdR incorporation) with IC50 values of 430 nM and 2.3 microM, respectively. These effects appeared to be specifically related to PDGF receptor tyrosine kinase inhibition since WIN 41662 was not cytotoxic (in vitro) and since WIN 72039, a close structural analog that does not inhibit PDGF receptor tyrosine kinase, also did not inhibit PDGF-stimulated receptor autophosphorylation, Ca2+ mobilization, or hVSMC proliferation. Thus, WIN 41662 is representative of a novel class of selective PDGF receptor tyrosine kinase inhibitors that inhibit PDGF-regulated secondary events in intact cells.

We report the synthesis and in vitro biological activity of the nonpeptide bradykinin receptor antagonist WIN 64338, [[4-[[2-[[bis(cyclohexylamino)methylene]amino]-3-(2- naphthyl)-1-oxopropyl]amino]phenyl]methyl]tributylphosphonium chloride monohydrochloride. WIN 64338 inhibits [3H]-bradykinin binding to the bradykinin B2 receptor on human IMR-90 cells with a binding inhibition constant (Ki) of 64 +/- 8 nM and demonstrates competitive inhibition of bradykinin-stimulated 45Ca2+ efflux from IMR-90 cells (pA2 = 7.1). The antagonist inhibits bradykinin-mediated guinea pig ileum contractility (pA2 = 8.2) and has significantly weaker activity against acetylcholine-induced contractility in the same preparation. WIN 64338 is not active in a rabbit aorta bradykinin B1 receptor assay, demonstrating that it is a selective bradykinin B2 receptor antagonist. The compound inhibits [3H]quinuclidinyl benzilate binding to the rat brain muscarinic receptor (Ki = 350 nM) but is 25- to 100-fold more selective for the bradykinin receptor compared with other receptors against which it has been tested. Synthesis of WIN 64338 has provided a nonpeptide competitive bradykinin B2 antagonist active in both bradykinin radioligand binding and functional assays.

D-Arg0[Hyp3,Thi5,D-Tic7,Oic8]bradykinin (HOE-140) is a potent (Ki = 0.11 nM) inhibitor of [3H]bradykinin binding to bradykinin B2 receptors found on human IMR-90 fetal lung fibroblasts. During the synthesis of this compound, we isolated and unambiguously identified the L-Tic7 stereoisomer (WIN 65365), which exhibits a 2000-fold lower binding affinity (Ki = 130 nM) than HOE-140 to the bradykinin receptor. A similar decrease in potency is observed for WIN 65365 inhibition of bradykinin-stimulated 45Ca2+ efflux from IMR-90 cells. Both HOE-140 and WIN 65365 appear to be competitive antagonists at the IMR-90 bradykinin receptor. This is the first documentation of bradykinin binding and functional antagonist activity by a bradykinin peptide analogue with an L amino acid replacing Pro7. In an attempt to rationalize the differences in binding affinities of HOE-140 and WIN 65365, a conformational analysis of the peptides was undertaken using annealed molecular dynamics (AMD). Conformational analysis of HOE-140 reveals a strong preference for the formation of a type II' beta-turn in the carboxy-terminal region. Analogous modeling of WIN 65365 reveals that its conformation is strikingly different from HOE-140 in that the four carboxy-terminal residues of WIN 65365 do not form a beta-turn. These differences in low-energy conformations between the two peptides may lead to a better understanding of the molecular interaction of antagonists with the bradykinin receptor.

In this study, we examined the effect of TNFa on bradykinin (BK) B 2 receptor binding and function in human A431 epidermoid carcinoma cells. [ 3 H]BK binds to a single class of receptors on A431 cells in a saturable and reversible manner. A binding affinity ( K D ) of 3.0 ± 0.3 nM ( n = 4) and a B max of 151 ± 14 fmols/10 6 cells, representing approximately 90,000 BK receptors per cell, was observed. The rank order of potency for BK agonist peptides indicates that the A431 BK receptor appears to be of the B 2 subtype. When A431 cells were incubated with TNFa (10 ng/ml) for 48 h prior to BK binding, a significant decrease in the number of BK receptors compared to control was observed. TNFa did not influence the affinity of BK binding to A431 cells and direct addition of TNFa to the binding assay did not effect BK binding. BK-stimulated IP 1 formation appeared to be increased in TNFa treated cells compared to control whereas histamine-stimulated IP 1 formation was not influenced. Both control and TNFa treated cells were greater than 95% viable. However, TNFa treated cells were blocked in the G 1 phase of the cell cycle resulting in a decrease in DNA synthesis. This may be one mechanism for the TNFa-induced decrease in BK receptors in A431 cells.

The authors previously described the production of four monoclonal antibodies to the ..beta..-adrenergic receptor antagonist alprenolol. One of these antibodies, 5B7 (IgG/sub 2a/, kappa), was used to raise anti-idiotypic antisera in rabbits. In contrast to the expected results, one of the anti-idiotypic antisera (R9) promotes (/sup 125/I)iodocyanopinodolol (ICYP) binding to antibody 5B7. In the presence of R9, the dissociation constant decreases 100-fold from 20 to 0.3 nM. This increase in binding affinity of antibody 5B7 for ICYP is not observed in the presence of preimmune, rabbit anti-mouse or anti-idiotypic antisera generated to a monoclonal antibody of a different specificity. Furthermore, R9 in the absence of 5B7 does not bind ICYP. The F(ab) fragments of 5B7 and T9 behaved in a similar manner, and the soluble complex responsible for the high-affinity interaction with ICYP can be identified by gel filtration chromatography. The elution position of the complex is consistent with a 5B7 F(ab)-R9 F(ab) dimer, indicating that polyvalency is not responsible for the enhanced ligand binding. Kinetic analysis of ICYP-5B7 binding revealed that the rate of ICYP dissociation from 5B7 in the presence of R9 is approximately 100 times slower than in the absence of R9, consistent with the 100-fold changemore » in binding affinity of 5B7 for ICYP. The available data best fit a model in which an anti-idiotypic antibody binds at or near the binding site of the idiotype participating in the formation of a hybrid ligand binding site. This would allow increased contact of the ligand with the idiotype-anti-idiotype complex and result in an enhanced affinity of the ligand interaction.« less

A number of imines (Schiff bases) have been prepared from ketosteroids (estrone, estrone-3-methyl ether, androsterone, epiandrosterone, 5α-cholestan-3-one, 6-ketocholestanol and testosterone) and primary amines (phenethylamine, tyramine and hexanamine). The CD spectra of these compounds have been measured in methanol. Except for the testosterone imines, the azomethine n-π* band (~ 235 nm) exhibited the same sign (but not as high an ellipticity) as the parent ketone's n - π* band (~295 nm). The imines of the estrogens (estrone and estrone 3-methyl ether) gave CD spectra characterized by a negative 1Lb Cotton effect ([θ] ⋍ -1000) near 280 nm and a band near 230 nm ([θ] ⋍ +19,000) which is considered the sum of a positive azomethine n - π* Cotton effect and positive aromatic 1La Cotton effect(s). The 1La band originating in the amine moiety was prominent in the CD of 5α-cholestan-3-one and 6-ketocholestanol phenethylamine imines. When aromatic Cotton effects originating in the amine moiety were detected, they were of the same sign as the n - π* Cotton effect.

The human promyelocytic cell line HL-60, differentiates in response to a variety of agents including dibutyrl cAMP and agents which increase intracellular cAMP concentrations (phosphodiesterase inhibitors, PGE 2 , and cholera toxin). HL-60 is also known to be rich in H 2 -hhistamine sensitive adenylate cyclase activity. The present study was therefore designed to test the effects of H 2 - stimulation on growth and differentiation of HL-60 using the potent H 2 agonist dimaprit. Dimaprit markedly increased cAMP production in a dose-dependent manner reaching maximal levels after 30–60 minutes. Intracellular cAMP levels decreased thereafter and by 24 hours were approximately 2–3 fold increased above control. Intracellular cAMP levels were not altered by dimaprit (10 −7 M to 10 −4 M) at 4 days in culture compared to either untreated HL-60 cells or dimethylsulfoxide (DMSO) (1.3%) treated cells. While exponential growth was unaltered by dimaprit (10 −7 M to 10 −4 M) as compared to control, dimaprit induced i) morphologic maturation to the myelocyte and metamyelocyte form with no differentiation seen beyond the metamyelocyte even after 6 days in culture, ii) increased NBT reductase activity and iii) dose-dependent increase in lysozyme activity which could be completely blocked by cimetidine, a specific H 2 antagonist. Dimaprit-induced differentiation of HL-60 cells was associated with an initial but transient increase in intracellular cAMP production. Maturation beyond the metamyelocyte stage was not observed. Acquisition of NBT reductase and lysozyme activity correlated with morphologic maturation.

A previous report demonstrated both immunological crossreactivity and structural similarity between the mammalian beta adrenergic receptor and the cell surface receptor for the reovirus type 3 (14). We now demonstrate that reovirus type 3 can bind selectively and with high affinity to cells that lack beta adrenergic receptor activity (L-cells). The present study was also designed to determine what effect reovirus binding has on beta adrenergic receptor function in cells (DDT1) that possess an intact ligand binding site. Based on computer analysis of reovirus competitive inhibition curves, the apparent dissociation binding constants (Kd) for reovirus binding to DDT1 and L-cells are 0.1 nM and 0.25 nM, respectively. High affinity [125I]-iodocyanopindolol (CYP) binding to beta adrenergic receptors can also be demonstrated in DDT1 cells but not in L-cells. In agreement with these ligand binding studies, adenylate cyclase activity is stimulated by the beta receptor agonist isoproterenol in DDT1 cell membranes but not in L-cell membranes. In addition, isoproterenol increases cAMP levels in DDT1 cells but not in L-cells. Neither reovirus serotype stimulates cAMP levels in either cell line, nor do they influence beta-adrenergic agonist stimulation of cAMP in DDT1 cells. These results argue against identity of the receptors for reovirus type 3 and beta adrenergic ligands.

The β-adrenergic receptor has been one of the most thoroughly studied membrane proteins to date. Interest in the receptor stems primarily from its role in the regulation of cardiovascular function at all levels. As a result, there have been many approaches to the study and characterization of the receptor polypeptide and the elucidation of how the receptor interacts with other membrane components. A novel method, the use of anti- idiotypic antibodies raised against antibodies specific for β -adrenergic receptor ligands, affords an immunological approach to the problem. Here we describe some pharmacological and physiochemical features of the β-adrenergic receptor and discuss some of the relevant literature on applications of immunological techniques to the study of the receptor at the molecular level.

At rat hepatic membrane alpha 1-adrenergic receptors, the nonhydrolyzable GTP analogue p[NH]ppG causes a rightward shift of agonist competition curves and a loss of high-affinity binding. This p[NH]ppG effect is consistent with the involvement of a guanine nucleotide-binding regulatory protein (G-protein) in alpha 1-adrenergic receptor signalling. Although readily apparent in membranes prepared to avoid retention of endogenous nucleotides and activation of Ca2+-sensitive proteinases (+pi), this p[NH]ppG effect is not observed in membranes prepared without proteinase inhibitors (-pi), or in -pi membranes treated with Ca2+ (-pi, +Ca2+). In these various membrane preparations, different Mr forms of the receptor are also identified by photoaffinity labeling with [125I]CP65526, an aryl azide analog of the alpha 1-selective antagonist, prazosin, followed by SDS-polyacrylamide gel electrophoresis and autoradiography. Whereas a predominant Mr = 80,000 subunit is identified in +pi membranes, in -pi membranes a proteolytic Mr = 59,000 fragment is also observed. In -pi, +Ca2+ membranes, only this latter peptide is detected. To evaluate the ability of each of these forms of the receptor to couple with a G-protein, the effect of p[NH]ppG on the agonist-inhibition of [125I]CP65526 labelling was determined by laser densitometry scanning and computer analysis. At the Mr = 80,000 subunit, p[NH]ppG causes a rightward shift of agonist competition curves and a loss of high-affinity binding, even in -pi membranes. By contrast, agonist-binding at the Mr = 59,000 subunit is of low-affinity and was not affected by p[NH]ppG. These data indicate that the cleaved Mr = 59,000 fragment, while retaining hormone binding activity is unable to undergo G-protein coupling. Thus, the alpha 1-adrenergic receptor appears to contain a discrete domain necessary for G-protein coupling that is distinct from its ligand recognition site.

Improved methods for the solubilization and purification of the mammalian beta 2-adrenergic receptor have allowed this protein to be characterized further. In the present study, the beta 2-adrenergic receptor has been solubilized from calf lung membranes using a 0.4% digitonin/0.08% cholate-Tris buffer with multiple proteinase inhibitors. This solubilization buffer produced 60-75% solubilization of the receptor, which retained complete ligand-binding activity as determined by Scatchard analysis. Subsequent receptor purification employed a modified acebutolol-agarose affinity resin. The eluate from the affinity resin was then purified further by HPLC-gel exclusion chromatography on a Spherogel TSK-3000 column. The receptor, detected by [3H]dihydroalprenolol or [125I]iodocyanopindolol binding, eluted with a retention time identical to that of IgG (Stokes radius 49 A). Autoradiography following SDS-PAGE of the purified iodinated receptor clearly demonstrated two distinct bands: a major band of 67 kDa and a minor band of 53 kDa. With the addition of leupeptin to the proteinase inhibitor regimen, the 53-kDa band became less apparent. Two-dimensional gel electrophoresis indicated that the 67-kDa peptide behaved as a predominantly single species with a pI of 6.0 +/- 0.2. The purified receptor protein recognized adrenergic ligands with a specificity identical to that of the membrane-bound beta 2-adrenergic receptor.