Frog Erythrocyte Membranes Research Articles

Kinetics of K-Cl cotransport in frog erythrocyte membrane: effect of external sodium.

In frog red blood cells, K-Cl cotransport (i.e., the difference between ouabain-resistant K fluxes in Cl and NO(3)) has been shown to mediate a large fraction of the total K(+) transport. In the present study, Cl(-)-dependent and Cl(-)-independent K(+) fluxes via frog erythrocyte membranes were investigated as a function of external and internal K(+) ([K(+)](e) and [K(+)](i)) concentration. The dependence of ouabain-resistant Cl(-)-dependent K(+) ((86)Rb) influx on [K(+)](e) over the range 0-20 mm fitted the Michaelis-Menten equation, with an apparent affinity (K(m)) of 8.2 +/- 1.3 mm and maximal velocity (V(max)) of 10.4 +/- 1.6 mmol/l cells/hr under isotonic conditions. Hypotonic stimulation of the Cl(-)-dependent K(+) influx increased both K(m) (12.8 +/- 1.7 mm, P < 0.05) and V(max) (20.2 +/- 2.9 mmol/l/hr, P < 0.001). Raising [K(+)](e) above 20 mm in isotonic media significantly reduced the Cl(-)-dependent K(+) influx due to a reciprocal decrease of the external Na(+) ([Na(+)](e)) concentration below 50 mm. Replacing [Na(+)](e) by NMDG(+) markedly decreased V(max) (3.2 +/- 0.7 mmol/l/hr, P < 0.001) and increased K(m) (15.7 +/- 2.1 mm, P < 0.03) of Cl(-)-dependent K(+) influx. Moreover, NMDG(+) Cl substitution for NaCl in isotonic and hypotonic media containing 10 mm RbCl significantly reduced both Rb(+) uptake and K(+) loss from red cells. Cell swelling did not affect the Na(+)-dependent changes in Rb(+) uptake and K(+) loss. In a nominally K(+)(Rb(+))-free medium, net K(+) loss was reduced after lowering [Na(+)](e) below 50 mm. These results indicate that over 50 mm [Na(+)](e) is required for complete activation of the K-Cl cotransporter. In nystatin-pretreated cells with various intracellular K(+), Cl(-)-dependent K(+) loss in K(+)-free media was a linear function of [K(+)](i), with a rate constant of 0.11 +/- 0.01 and 0.18 +/- 0.008 hr(-1) (P < 0.001) in isotonic and hypotonic media, respectively. Thus K-Cl cotransport in frog erythrocytes exhibits a strong asymmetry with respect to transported K(+) ions. The residual, ouabain-resistant K(+) fluxes in NO(3) were only 5-10% of the total and were well fitted to linear regressions. The rate constants for the residual influxes were not different from those for K(+) effluxes in isotonic ( approximately 0. 014 hr(-1)) and hypotonic ( approximately 0.022 hr(-1)) media, but cell swelling resulted in a significant increase in the rate constants.

Temperature effects on ion transport across the erythrocyte membrane of the frog Rana temporaria.

Unidirectional K+ and Na+ influxes in the frog erythrocytes incubated in Cl- or NO(3)- media with 2.7 mM K+ were measured using 86Rb and 22Na as tracers. K+ influx was inhibited by 35-55% in the presence of 0.2-1.0 mM furosemide but it was unaffected by 0.1-0.2 mM bumetanide. Furosemide at a concentration of 0.5 mM had no effect on K+ loss from the frog red cells incubated in a nominally K(+)-free medium. Together with our previous studies the data support the existence of K-Cl cotransport and the absence of Na-K-2Cl cotransport in the frog erythrocyte membrane. Cell cooling from 20 to 5 degrees C caused a decrease in K+ influx and K+ efflux via the K-Cl cotransporter (3.2- and 3.7-fold, respectively) giving an apparent energy of activation (EA) of about 60 kJ/mol and Q10 value of 2.5. Only small decline (approximately 30%) in the ouabain-sensitive K+ influx was found as temperature was changed from 20 to 5-10 degrees C. Low values of Q10 (approximately 1.5) and EA (27.3 kJ/mol) were obtained for passive K+ influx in the frog erythrocytes (ouabain-insensitive in NO(3)- medium) at temperature within 5-20 degrees C. However, the temperature coefficients were greater for passive Na+ influx and passive K+ efflux (Q10 approximately 2.4-2.5 and EA approximately 56-58 kJ/mol). The temperature dependence of all ion transport components displayed discontinuities showing no changes at temperature between 5 and 10 degrees C. Thus, cooling of the frog red cells is associated with a greater decrease of Na+ influx and K+ efflux than passive and active K+ influx. These data indicate that the preservation of a relative high activity of the Na,K-pump during cell cooling and also the temperature-induced changes in the K-Cl cotransport activity and ion passive diffusion contribute to maintenance of ion concentration gradients in the frog erythrocytes at decreased temperature.

Activation of the na +$z.sbnd;-k + pump in frog erythrocytes by catecholamines and phosphodiesterase blockers

K + and Na + influx into frog erythrocytes incubated in standard saline was studied using 86Rb and 22Na as tracers. 10 μM isoproterenol (1SP) produced a significant increase in K + influx for the first 15 min, which was sustained during the entire 60 min of cell incubation. Treatment of red cells with the phosphodiesterase (PDE) blockers theophylline (THEO, 1 and 5 mM) or 3-isobutyl-l-methylxanthine (IBMX, 0.5 mM) was also accompanied by an enhancement in K + influx. A distinct additive effect on K + influx into red cells was found when ISP and THEO or IBMX were added together. The increase in K + transport induced by ISP plus IBMX was totally abolished by pretreatment of red cells with 0.1 mM ouabain. The ouabain-sensitive K + influx in frog erythrocytes was elevated in the presence of ISP plus IBMX to 2.05 ± 0.45, as compared with the control level of 0.39 ± 0.11 mmol/L cells/hr ( P < 0.001). Similar effects of ISP and IBMX on K + influx were observed after chloride was replaced by nitrate. A dose-related increase in K + influx into frog erythrocytes was observed at ISP concentrations of 10 −8–10 −6 M, with a half-maximal stimulatory concentration of approximately 0.02 μ.M. The effects of ISP (10 −8–10 −58 M) on K + transport were completely abolished with 10 μM of the βadrenergic blocker propranolol, but α-adrenergic antagonists (phentolamine, prazosin, and yohimbine) did not alter the ISP-induced increase in K + influx. The drugs tested had no effect on 22Na influx in frog red cells, but ISP produced a small decline (13%) in intracellular Na + concentration. Thus, our study indicates that catecholamines and PDE blockers enhance K + ( 86Rb) transport in frog erythrocytes mediated by Na +-K + pump activity. The frog erythrocyte membrane may serve as a convenient model to investigate the hormonal modulation of the Na +-K + pump.

On the Contributions of R [formula omitted]Complex and Agonist-Receptor Complex to Formation of the Ternary Complex: Results of a New Method of Analysis

More than 2 decades ago it was demonstrated that responses of some well-studied biochemical systems were not proportional to occupation of receptors by hormones specific for the receptors, even when the receptors were considered to be very closely coupled to the response. A plausible hypothesis devised to account for some of those results involved the formation of a ternary complex consisting of receptor Rhormone (or transmitter) ligand Land so-called regulatory or transducer protein X . The most successful experimental-theoretical study of such a system was that of DeLean et al.(1980) who showed that the adenylate cyclase-linked β-adrenergic system of frog erythrocyte membranes conformed closely to the theoretical model of Jacobs & Cuatrecasas (1976). Not only did conformity to the theory require the formation of ternary complex X ̄ but also of significant quantities of binary complex X ̄ X ̄ as well. Black & Leff (1983) were the first to adapt the ternary complex hypothesis to describe experiments of the types commonly carried out by pharmacologists. It was later utilized for this purpose by others such as Mackay (1987, 1988), Morgan et al.(1988), Jenkinson (1989) and Leff & Harper (1989). Although all of these writers gave due attention to the ternary complex in their formulations, they omitted from their calculations the X ̄ complex to whose significance DeLean et al.had drawn attention. In the following treatment we have reconsidered the role of the binary X ̄ complex from new points of view and have shown that R X ̄ complex contributed to formation of the ternary complex in the experiments of DeLean et al.but that agonist-receptor complex did not do so. These results contradict the suggestion of DeLean et al.concerning the “simplest” mechanistic explanation of their observations.

Changes in immunohistochemical properties of beta-adrenergic receptors in frog erythrocytes by isoproterenol-induced desensitization

Immunohistochemical properties of beta-adrenergic receptor (BAR) in frog erythrocytes have been studied by using antiserum raised against purified guinea pig BAR. Immunoblotting of frog erythrocyte membranes with the anti-BAR serum revealed prominent staining of a protein with Mr of 65,000–67,000. BARs present in intact frog erythrocytes were made visible by incubation with the anti-BAR serum and then goat-anti rabbit IgG conjugated with colloidal gold. About 50–60% of the cells showed small, punctate dots by staining with the anti-BAR serum. After 4 hr exposure of the cells to isoproterenol, the density of the staining was markedly increased. Labeling of BAR after permeabilization of erythrocytes with saponin was markedly enhanced in isoproterenol-desensitized, but not in control cells. The BARs present in cytoslic fraction of desensitized cells migrated in the void volume of Sepharose-4B and were effectively labeled by a lipophilic BAR ligand capable of penetrating the cell membranes, but not by a hydrophilic ligand. Thus, isoproterenol-induced desensitization is associated with alteration of the immunoreactivity of BAR. Moreover, our immunochemical and biochemical data provide further evidence for the internalization of BAR in desensitized frog erythrocytes.

Quantitative structure-activity relationships of beta-adrenergic agents. Application of the computer automated structure evaluation (CASE) technique of molecular fragment recognition

A quantitative structure-activity analysis of adenylate cyclase-coupled beta-adrenergic receptor agonists and antagonists in the frog erythrocyte membrane was made. On the basis of molecular structural fragment descriptors, automatically generated by the CASE methodology, catecholamine derivatives were correctly classified as agonists or antagonists. The potency of these agents in each category, as well as their binding-affinity for the beta receptor was correlated through a multivariate regression analysis.

Phorbol diester treatment promotes enhanced adenylate cyclase activity in frog erythrocytes

Incubation of intact frog erythrocytes with 12- O-tetradecanoyl phorbol-13-acetate (TPA), a tumor-promoting phorbol diester which activates protein kinase C, results in an approximate two- to threefold increase in subsequently tested β-adrenergic agonist-stimulated adenylate cyclase activity. This increase is due to an elevation in the V max of the enzyme rather than to a change in affinity for the agonist. TPA treatment of frog erythrocytes does not alter the affinity ( K D ) or the binding capacity ( B max) for the β-adrenergic antagonist [ 125I]cyanopindolol. In addition, agonist/[ 125I]cyanopindolol competition curves are not affected by TPA pretreatment nor is their sensitivity to guanine nucleotides. Incubation of frog erythrocyte membranes alone with TPA does not promote sensitization or activation of adenylate cyclase activity. Pretreatment of intact frog erythrocytes with TPA also produces approximately two- to threefold increases in basal, guanine nucleotide-, prostaglandin E 1-, forskolin-, NaF-, and MnCl 2-stimulated adenylate cyclase activities in frog erythrocyte membranes. This enhancement of adenylate cyclase activity by TPA is induced rapidly ( t 1 2 ≅ 5 min ) and with an EC 50 of about 10 −7 to 10 −6 m. Other tumor-promoting phorbol diesters or phorbol diester-like compounds including 4β-phorbol 12,13-dibutyrate, 4β-phorbol 12,13-didecanoate, and mezerein are effective in promoting enhanced adenylate cyclase activity. In contrast, phorbols such as 4β-phorbol, 4α-phorbol 12,13-didecanoate, and 4- O-methylphorbol 12-myristate 13-acetate, which are inactive in tumor promotion and which do not activate protein kinase C, do not affect frog erythrocyte adenylate cyclase activity. These data are suggestive of a protein kinase C-mediated phosphorylation of one of the adenylate cyclase components that is distal to the receptor, i.e., the nucleotide regulatory and/or catalytic components.

Sensitization of frog erythrocyte adenylate cyclase system by tumor-promoting phorbol diesters

Preincubation of frog erythrocyte lysates with tumor-promoting phorbol diesters leads to an increase in adenylate cyclase activity. This stimulatory effect of phorbol diesters was specific. Incubation with 12-O-tetradecanoylphorbol 13-acetate led to increases in basal (38%) and isoproterenol- (40%), fluoride- (25%), and Mn-stimulated (68%) adenylate cyclase activities compared with control. The inactive phorbol diesters (4 alpha-phorbol 12,13-didecanoate and beta-phorbol) were ineffective in promoting increases in adenylate cyclase activity. The effect of active phorbol diesters was also observed on isolated frog erythrocyte membranes in the absence of cell supernatant, although to a much lesser extent than in the whole lysates. Addition of the cell supernatant or of purified protein kinase C to the membranes maximized the sensitization by the phorbol diesters. These data are consistent with the notion that some component(s) of the adenylate cyclase system is (are) phosphorylated by protein kinase C, resulting in an enhancement of enzyme activity.

Mapping of mammalian β-adrenoreceptors by use of macromolecular alprenolol derivatives: A comparison with amphibian erythrocyte receptors

The interaction of macromolecular alprenolol derivatives with β-adrenoreeeptors of rat heart, lung, and erythrocytes and frog erythrocytes has been studied. Macromolecular derivatives were prepared by covalently coupling alprenolol to dextrans containing a homologous series of spacer arms of various lengths. The affinity of these macromolecules for frog erythrocyte membranes increased with increasing length of spacer arm. In contrast, the affinity of these macromolecules for all mammalian membrane preparations was weak and insensitive to the length of the spacer arm. The inhibition of [ 3H]dihydroalprenolol binding to rat heart, lung, and erythrocyte membrane preparations by these macromolecular derivatives was more than 1000-fold less potent than inhibition by alprenolol. The results suggest different structural characteristics between mammalian and amphibian β-adrenoreceptors; however, apparently only small differences between mammalian receptors could be distinguished with these probes.

The beta-adrenergic receptor: rapid purification and covalent labeling by photoaffinity crosslinking.

New procedures for the rapid purification and covalent labeling of the beta-adrenergic receptors have been developed that should greatly accelerate progress in the study of these widely distributed adenylate cyclase-coupled receptors. Chromatography of solubilized receptor preparations on a Sepharose-alprenolol affinity gel followed by HPLC on steric exclusion columns lead to rapid (2 days) and high yield (approximately 30%) purification of the receptors from frog erythrocytes. The receptor obtained by these rapid procedures appears to be composed entirely of 58,000 Mr subunit(s) and to be identical to that previously purified by much lengthier procedures [Shorr, R. G. L., Lefkowitz, R. J. & Caron, M. G. (1981) J. Biol. Chem. 256, 5820-5826]. A novel, very high affinity, specific beta-adrenergic antagonist, p-aminobenzylcarazolol, has also been synthesized. It can be radioiodinated to theoretical specific radioactivity with 125I (2,200 Ci/mmol). This radioligand, which possesses an arylamine moiety, may then be covalently incorporated into the receptor binding subunit (58,000 Mr peptide) of the frog erythrocyte membranes by the use of the bifunctional photoactive crosslinker N-succinimidyl-6-(4'-azido-2'- nitrophenylamino)hexanoate (SANAH). Covalent incorporation is blocked by various drugs with a strict beta-adrenergic specificity. This suggests that the photoaffinity crosslinking approach may be useful for labeling a variety of small molecule and neurotransmitter receptors when appropriate ligands can be synthesized.

Photoaffinity labeling of the beta-adrenergic receptor.

A new photoactive beta-adrenergic antagonist, p-azidobenzylcarazolol (pABC) has been synthesized by combining a carbazole moiety with a p-azido-benzyl substituent. The compound has been labeled with tritium to a specific activity of 26 Ci/mmol. In frog erythrocyte membranes, [3H]p-azido-benzylcarazolol binds to the beta-adrenergic receptor with the expected beta 2 specificity and with high affinity (KD congruent to 100 +/- 10 pM). Unlabeled p-azido-benzylcarazolol can irreversibly inactivate the [3H]dihydroalprenolol-binding activity of frog erythrocyte membranes in a photodependent manner which can be prevented by beta-adrenergic agents. Incubation of frog erythrocyte membranes or digitonin-solubilized preparations of these membranes or digitonin-solubilized preparations of these membranes which had been enriched in beta-adrenergic receptors by a Sepharose-alprenolol chromatography step led to covalent incorporation of radioactivity into a Mr = 58,000 peptide. Specific incorporation of [3H]pABC into the Mr = 58,000 peptide could be prevented by both beta-adrenergic agonists and antagonists. This peptide has previously been purified and shown to contain the beta-adrenergic receptor-binding site (Shorr, R. G. L., Lefkowitz, R. J., and Caron, M. G. (1981) J. Biol. Chem. 256, 5820-5826). Thus, photoaffinity labeling of the beta-adrenergic receptor protein directly identifies the same hormone-binding subunit as has been isolated by conventional purification techniques.

Evidence that a beta-adrenergic receptor-associated guanine nucleotide regulatory protein conveys guanosine 5'-O-(3-thiotriphosphate)- dependent adenylate cyclase activity.

The guanine nucleotide regulatory protein component (N) of the frog erythrocyte membrane adenylate cyclase system appears to form a stable complex with the beta-adrenergic receptor (R) in the presence of agonist (H). This agonist-promoted ternary complex HRN can be solubilized with Lubrol. The guanine nucleotide regulatory protein associated with the solubilized complex can be adsorbed either to GTP-Sepharose directly or to wheat germ lectin-Sepharose via its interaction with the receptor which is a glycoprotein. Guanosine 5'-O-(3-thiotriphosphate)(GTP gamma S) can be used to elute the guanine nucleotide regulatory protein from either Sepharose derivative. The resulting N.GTP gamma S complex conveys nucleotide-dependent adenylate cyclase activity when combined with a Lubrol-solubilized extract of turkey erythrocyte membranes. The ability to observe GTP gamma S-dependent reconstitution of adenylate cyclase activity in the eluate from either resin required the formation of the HRN complex prior to solubilization. The N protein can be identified by its specific [32P]ADP ribosylation catalyzed by cholera toxin in the presence of [32P]NAD+. The existence of a stable HRN intermediate complex is supported by the observation that agonist pretreatment of frog erythrocyte membranes results in a 100% increase in the amount of 32P-labeled N protein eluted from the lectin-Sepharose in the presence of GTP gamma S compared to membranes pretreated with either antagonist or agonist plus GTP. Our results therefore provide evidence that the same guanine nucleotide-binding protein that associates with the beta-adrenergic receptor in the presence of agonist mediates adenylate cyclase activation.

Correlation of beta-adrenergic receptor-stimulated [3H]GDP release and adenylate cyclase activation. Differences between frog and turkey erythrocyte membranes.

Correlation of beta-adrenergic receptor-stimulated [3H]GDP release and adenylate cyclase activation. Differences between frog and turkey erythrocyte membranes.

Cytoskeletal constraint of the beta-adrenergic receptor in frog erythrocyte membranes.

A fluorescence receptor binding assay, based upon the high-affinity beta-adrenergic receptor antagonist propranolol, is utilized to probe the microenvironment of the antagonist-receptor complex in the frog (Rana catesbeiana) erythrocyte membrane. The technique of steady-state fluorescence depolarization is applied to the propranolol-receptor complex, allowing quantitation of the rotational relaxation time of the complex. It is found that the complex is dynamically constrained at 20 degrees C. However, in the temperature range 6-10 degrees C a sharp reversible release of constraint is observed. It is further demonstrated that the addition of drugs that are known to specifically disrupt the cytoskeleton (colchicine, vincristine, and vinblastine) causes a similar but irreversible release of constraint at 20 degrees C. Cytochalasin B has a much smaller influence on the rotational mobility of the propranolol-receptor complex than do the other drugs that disrupt the cytoskeleton. Amphotericin B is without effect on the rotational constraint of the complex. Binding of the antagonist [3H]dihydroalprenolol is not influenced by colchicine. A model is proposed which postulates that cytoskeletal elements are linked to the antagonist-receptor complex. Antagonist binding does not result in cytoskeletal release, whereas agonist binding is postulated to lead to dissociation of the agonist-receptor complex from the cytoskeleton, thereby activating adenylate cyclase.

Effects of local anesthetics of guanyl nucleotide modulation of the catecholamine-sensitive adenylate cyclase system and on β-adrenergic receptors

Tetracaine and other local anesthetics exert multiple actions on the catecholamine-sensitive adenylate cyclase system of frog erythrocyte membranes. Tetracaine (0.2–2.0 mM) reduces the responsiveness of adenylate cyclose to (a) guanyl-5′-yl-imidodiphosphate and (b) isoproterenol in the presence of GTP or guanyl-5′-yl-imidodiphosphate. Local anesthetics did not affect (a) basal enzyme activity, and (b) enzyme responsiveness to NaF. Tetracaine inhibited stimulation of adenylate cyclase by guanyl-5′-yl-imidodiphosphate over the whole range of nucleotide concentrations. By contrast, inhibition by tetracaine of isoproterenol activity in the presence of GTP was significant only if GTP concentrations exceeded 10 −7 M. Tetracaine also competitively inhibited binding of both the antagonist [ 3H]-dihydroalprenolol and the agonist [ 3H]hydroxybenzylisoproterenol to β-adrenergic receptors. However, it was twice as potent in inhibiting [ 3H]-hydroxybenzylisoproterenol as [ 3H]dihydroalprenolol binding. The greater potency for inhibition of agonist binding was due to the ability of the anesthetics to promote dissociation of the high-affinity nucleotide sensitive state of the β-adrenergic receptor induced by agonists. Other local anesthetics mimicked the effects of tetracaine on adenylate

A high affinity agonist . beta-adrenergic receptor complex is an intermediate for catecholamine stimulation of adenylate cyclase in turkey and frog erythrocyte membranes.

A high affinity agonist . beta-adrenergic receptor complex is an intermediate for catecholamine stimulation of adenylate cyclase in turkey and frog erythrocyte membranes.

Loss of beta-adrenergic receptor-guanine nucleotide regulatory protein interactions accompanies decline in catecholamine responsiveness of adenylate cyclase in maturing rat erythrocytes.

The maturation of rat reticulocytes to erythrocytes is paralleled by a marked loss of adenylate cyclase responsiveness to catecholamines and guanine nucleotides without significant loss of basal enzyme activity. These changes in adenylate cyclase activity are accompanied by a parallel %fold decrease in both the number of p-adrenergic binding sites and the quantity of M, = 42,000 protein substrates for cholera toxin-catalyzed [32P]ADP ribosylation. In addition, the maturation of the rat erythrocyte is accompanied by a loss of the ability of guanine nucleotides to modulate receptoragonist interactions. Thus, whereas guanine nucleotides promote tritiated hydroxybenzylisoproterenol agonist dissociation from the P-adrenergic receptor and reduce the potency of isoproterenol in competing for dihydroalprenolol antagonist binding in rat reticulocyte membranes, these regulatory effects of guanine nucleotides are virtually absent in membranes derived from mature rat erythrocytes. Furthermore, the ability of agonist occupancy of the P-adrenergic receptor of rat reticulocyte membranes to promote an increase in receptor size, as assessed by gel exclusion chromatography of solubilized preparations, is not observed in mature erythrocyte preparations. This agonist-induced increase in apparent receptor size has been previously observed in frog erythrocyte membranes (Limbird, L. E., and Lefkowitz, R. J. (1978) Proc. NatZ. Acad. Sei. U. S. A. 75,228-232) and is thought to reflect the formation of a receptor guanine nucleotide regulatory complex. Consistent with this hypothesis is the ability of guanine nucleotides to reverse the formation of this larger molecular size form of the receptor in rat reticulocyte membranes. The inability to promote this receptorguanine nucleotide regulatory protein complex in rat erythrocyte membranes suggests that one molecular concomitant of rat erythrocyte maturation is a decrease in the effectiveness of receptor-guanine nucleotide regulatory protein communication.

Differential effects of GTP on the coupling of β-adrenergic receptors to adenylate cyclase from frog and turkey erythrocytes application of new methods for the analysis of receptor-effector coupling

A detailed comparison of the interaction of β-adrenergic receptors with adenylate cyclase stimulation and modification of this interaction by guanine nucleotides has been made in two model systems, the frog and turkey erythrocyte. Objective analysis of the data was facilitated by the development of new graphical methods which involve the use of logit-logit transformations of percent receptor occupancy versus percent enzyme stimulation plots (coupling curves). Receptor-cyclase coupling in turkey erythrocyte membranes demonstrates a proportional relationship between receptor occupancy and adenylate cyclase activation and is unaffected by exogenous guanine nucleotides. By comparison, the proportional relationship of receptor occupancy and adenylate cyclase activation observed in frog erythrocyte membranes in the absence of guanine nucleotides is modified by the addition of exogenous guanine nucleotides such that a greater fractional enzyme stimulation is elicited by low receptor occupancy. Methodological criteria crucial for valid comparison of receptor occupancy and adenylate cyclase activity are delineated. In addition, the possible molecular mechanisms of receptor-cyclase coupling which might give rise to the coupling curves observed are discussed.

Antibodies raised against purified beta-adrenergic receptors specifically bind beta-adrenergic ligands.

Antibodies raised against purified beta-adrenergic receptors themselves specifically bind beta-adrenergic ligands. Digitonin-solubilized frog (Rana pipiens) erythrocyte beta-adrenergic receptors, purified 100- to 200-fold by adsorption to an alprenolol-agarose affinity support and specifically eluted from the affinity resin by 1-100 mM (+/-)-isoproterenol, were used to immunize six rabbits. All immune sera, in contrast to preimmune sera, bound the beta-adrenergic antagonist [(3)H]Dihydroalprenolol binding activity was due to immunoglobulins. By competition studies, antibody [(3)H]dihydroalprenolol binding was found to display a specificity and stereoselectivity resembling that of the beta-adrenergic receptor, [i.e., (-)-isoproterenol > (-)-epinephrine > (-)-norepinephrine; alprenolol approximately propranolol >> phentolamine = aloperidol; and (-) isomers of both agonists and antagonists 10-100 times more potent than (+) isomers]. A portion of the [(3)H]dihydroalprenolol binding antibodies could be specifically adsorbed onto purified frog erythrocyte membranes, whereas Xenopus and human erythrocyte membranes, both of which are almost devoid of beta-adrenergic receptors, were ineffective in adsorbing [(3)H]dihydroalprenolol binding antibodies. We suggest that the likely immunogen was a beta-adrenergic receptor-isoproterenol complex and that immunization with drugs noncovalently bound to their receptors might be a means of raising antibodies to biologically active otherwise nonimmunogenic small molecules. Such antibodies, whose specificity mimics that of a receptor, should also provide useful models for the study of the structure of the receptor binding sites.

Agonist-induced increase in apparent β-adrenergic receptor size

The properties of digitonin-solubilized beta-adrenergic receptors from frog erythrocyte membranes were studied by gel exclusion chromatography on AcA 34 Ultragel. beta-Adrenergic receptor binding activity in these membranes can be identified by both an agonist ligand, [(3)H]hydroxybenzylisoproterenol, and the antagonist ligands, [(3)H]dihydroalprenolol and (125)I-labeled hydroxybenzylpindolol. Occupancy of the beta-adrenergic receptors with the [(3)H]hydroxybenzylisoproterenol agonist prior to their solubilization from the membrane leads to an increase in apparent receptor size. Alterations in the molecular size of the receptor cannot be mimicked by occupancy of the binding site with the antagonist ligands. Exposure of frog erythrocyte membranes to [(3)H]hydroxybenzylisoproterenol agonist in the presence of 10 muM Gpp(NH)(p), a guanyl nucleotide analog that exerts multiple regulatory effects on the catecholamine-sensitive adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); EC 4.6.1.1] system, results in the elution of the [(3)H]hydroxybenzylisoproterenol radioligand in both the region characteristic of the agonist-receptor complex and the region characteristic of the antagonist-receptor complex. The precise molecular interactions responsible for the agonist-induced increase in apparent beta-adrenergic receptor size are still unresolved. However, the low concentrations of agonist that are capable of altering apparent receptor size and the sensitivity of this effect to guanyl nucleotides suggest that these phenomena may be intimately involved in eliciting the physiological effects of beta-adrenergic catecholamines at the molecular level.