Frog Erythrocytes Research Articles

Activation, desensitization, and recycling of frog erythrocyte beta-adrenergic receptors. Differential perturbation by in situ trypsinization.

We have utilized limited in situ trypsinization of the adenylate cyclase-coupled beta-adrenergic receptor of frog erythrocytes to probe the processes of receptor activation, desensitization, and recycling. Treatment of intact erythrocytes with trypsin (1 mg/ml) for 1 h at 20 degrees C converts all the receptor peptides (identified by photoaffinity labeling with p-azido-125I-benzylcarazolol) from a Mr approximately 58,000 to a Mr approximately 40,000 species. Nonetheless, the trypsinized beta-adrenergic receptors bind agonists and antagonists with unaltered affinity and with no change in the number of binding sites. Moreover, the ability of the proteolyzed receptors to interact with the nucleotide regulatory protein to form a high affinity guanine nucleotide-sensitive state and to activate adenylate cyclase were also unaltered. However, upon exposure of intact cells to the agonist isoproterenol, trypsinized beta-adrenergic receptors were more rapidly and more completely cleared from the plasma membranes ("down-regulated") than untrypsinized receptors. Whereas down-regulated receptors from nontrypsinized cells appear to recycle to the cell surface after removal of the agonist, internalized trypsinized beta-adrenergic receptors do not recycle to the plasma membrane and appear to be degraded within the cell. Moreover, when internalized receptors, recovered in a light vesicle fraction, were fused with a heterologous adenylate cyclase system, untreated but not trypsinized receptors reconstituted catecholamine stimulation of the enzyme. These data suggest that the beta-adrenergic receptor contains a trypsin-sensitive site which is exposed on the outer surface of the plasma membrane. Proteolysis at this site releases a fragment which though not critically involved in either ligand binding or "effector coupling" might be important for anchoring the receptors in the plasma membrane. These data also suggest that in situ proteolysis of the receptors might serve as a physiological trigger for their internalization and degradation.

Simultaneous existence of hemolysins and hemagglutinins in the coelomic fluid and in the cocoon albumen of the earthworm Eisenia fetida andrei

1. 1. Both hemolysis and hemagglutination activities are simultaneously present in the coelomic fluid and the cocoon albumen of Eisenia fetida andrei. 2. 2. Hemolysin was evidenced using fresh erythrocytes when hemagglutinin is revealed only on glutaraldehyde stabilized erythrocytes. 3. 3. Human, sheep, rabbit, rat, badger, chicken and frog erythrocytes were sensible to the coelomic fluid activities. 4. 4. The fractions containing the active molecules have been isolated by chromatography: one fraction possessed the 2 activities when another fraction only agglutinated. 5. 5. The possible involvement of these activities in antibacterial, morphogenetic and trophic functions is discussed.

Erythrocytic organic phosphates: Diel and seasonal cycles in the frog, Rana berlandieri

1. 1. Hemoglobin (Hb) concentration, hematocrit (Hct). total organic phosphates (P tot), nucleoside triphosphates (NTP), 2,3-diphosphoglycerate (DPG). and “inositol polyphosphates” (IPP) were measured in the erythrocytes of adult frogs at different times of day in winter and summer after acclimatization to 15 C and an LD 12:12 photoperiod. The same measurements were also made on animals acclimated to LD 16:8 in summer. 2. 2. All of the measured parameters varied significantly at different times of the day and between seasons in animals acclimated to an LD12:12 photoperiod. 3. 3. Summer animals acclimated to LD 16:8 showed significant diel cycles only in Hct and Hb.

Copper and zinc superoxide dismutase from the liver of bullfrog Rana catesbeiana

1. 1. A copper-zinc superoxide dismutase was purified to electrophoretic homogeneity from the liver of bullfrog, Rana catesbeiana. The enzyme has a mol. wt of 32,000 and is composed of two identical subunits, each containing one copper and one zinc atom. 2. 2. The amino acid composition of the enzyme differs in the contents of glutamic acid, glycine, alanine and valine from that of the enzymes from other species. The N-terminal amino acid is methionine. The sequence of 24 residues in the N-terminal region shows a good homology with that of the human, bovine and yeast enzymes. 3. 3. As judged by a double immunodiffusion test with the antibody prepared against the frog liver enzyme, the enzymes of the frog erythrocyte and tadpole liver and erythrocyte are immunologically the same as that of the frog liver enzyme.

Beta-adrenergic receptor internalization and processing: role of transglutaminase and lysosomes.

Membrane-bound β-adrenergic receptors are linked to adenylate cyclase using a guanine nucleotide binding protein as the linkage (1). It is well known that an extensive stimulation of these β-adrenergic receptors by β-receptor agonists attenuates the response of adenylate cyclase to further receptor stimulation — an event variously termed desensitization, subsensitivity, or down-regulation. In isolated frog erythrocytes (1, 2) and other systems (3–7), desensitization of β-adrenergic receptors is associated with a reduction in the number of membrane-bound receptor sites, suggesting that the density of surface receptor binding sites may control the magnitude of the receptor response.

Modifications of some erythrocyte parameters in hibernating vertebrates, revealed by cytofluorometric techniques

AbstractThe variations of the glycoconjugate concentrations (PAS‐positive material), with special reference to the membrane sialic component have been studied during the annual cycle in erythrocytes of three mammalian species (Erinaceus europaeus, Glis glis, Talpa europaea) and of one amphibian species (Rana esculenta). In R. esculenta the structural modifications of nuclear chromatin (different degree of compactness) have also been examined through the propidium iodide intercalation at different concentrations.The membrane sialic components during hibernation increase in Rana (457%), Glis (304%), and Erinaceus (244%), and decrease in Talpa (59%), not really a hibernating species. These data can be related with a slowing down of erythropoiesis and with the increase of the average length of erythrocyte life in the winter. Residual glycoconjugate components are also modified with a similar, but less evident, pattern. The changes in the fluorescence fading during the annual cycle show a substantial independence from the concentration index of the PAS‐positive material; this fact might be related to molecular modifications or heterogeneity of the substrate.The DNA‐propidium iodide intercalation in frog erythrocyte chromatin decreases during the hibernation, possibly related to a reduced functional activity of cells.

Pure beta-adrenergic receptor: the single polypeptide confers catecholamine responsiveness to adenylate cyclase.

The beta-adrenergic receptor binding subunits from frog erythrocytes, hamster lung and guinea pig lung have been purified to apparent homogeneity and in all cases reside on a single polypeptide. Insertion of the pure receptors into phospholipid vesicles and subsequent fusion of these vesicles with a receptor-deficient cell conveys beta-adrenergic responsiveness to the adenylate cyclase system of the acceptor cell. Such responsiveness is linearly dependent on the amount of receptor used in the fusion experiments and is independent of the receptor source. Moreover, this responsiveness displays appropriate beta-adrenergic specificity. These results indicate that the beta-adrenergic receptor polypeptide contains both the ligand binding site and the site responsible for mediating stimulation of adenylate cyclase activity, presumably via interaction with the guanine nucleotide regulatory protein.

Activation of adenylate cyclase by beta-adrenergic receptors: Investigation of rate limiting steps by simultaneous assay of high affinity agonist binding and GDP release

We report the development and application of a novel assay for high affinity binding of the agonist [ 3H]hydrozybenzyl-isoproterenol simultaneously with the agonist-promoted release of membrane bound [ 32P] GDP in the frog erythrocyte beta-adrenergic receptor system. We find that under various assay conditions both events occur with the same rate, ranging from 0.05 to 0.5 min −1. Addition of the non-hydrolyzable guanine nucleotide, guanylyl-imidodiphosphate simultaneously increases the rate of high affinity agonist binding and agonist promoted GDP release. In addition, the guanine nucleotide analog decreases the steady state level of high affinity agonist binding and increases the steady state level of agonist promoted GDP release with comparable potencies of 0.5 μM and 0.1 μM, respectively. The decrement in the steady state level of high affinity agonist binding (180 fmol/mg protein) due to the guanine nucleotide analog is in the same range as the reciprocal increment in the extent of agonist-induced [ 32P] GDP release (180 fmol/mg protein). The concommittant activation of adenylate cyclase, by submaximal concentrations of the agonist [ 3H]hydroxybenzylisoproterenol and guanylylimido-diphosphate under similar assay conditions proceeds with the same rate as for the two other measured functions of the system, i.e. 3H-agonist binding and agonist-promoted [ 32P] GDP release. This represents the first attempt at comparing the time course of adenylate cyclase activation with that of agonist binding and GDP release under similar assay conditions. The results indicate that GDP is not released prior to but rather coincident with formation of the complex of the hormone receptor with the regulatory protein and that enzyme activation proceeds with the same time course as agonist binds to the receptor. It is concluded that both high affinity agonist binding and GDP release represent integral aspects of the rate limiting step in the enzyme activation mechanism.

Reconstitution of beta-adrenergic receptors in lipid vesicles: affinity chromatography-purified receptors confer catecholamine responsiveness on a heterologous adenylate cyclase system.

The binding function of purified receptors can be assessed with radioligands, but the interaction of receptors with their biochemical effectors has not been amenable to direct study. Toward this end, procedures have been developed for directly demonstrating functionality of purified beta-adrenergic receptor preparations. Digitonin-solubilized beta-adrenergic receptors from frog erythrocytes or rat lung were purified approximately equal to 100- to 5,000-fold by affinity chromatography and inserted into a mixture of frog erythrocyte lipids and dimyristoyl phosphatidylcholine in the presence of octyl glucoside. Reconstitution of beta-adrenergic receptor binding was typically 25-50% and could also be effected with soybean phosphatidylcholine in the presence of octyl glucoside. The reconstituted beta-adrenergic receptors were then fused with Xenopus laevis erythrocytes, which contain prostaglandin E1-sensitive adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] but few beta-adrenergic receptors and little or no catecholamine-sensitive adenylate cyclase. Fusion of reconstituted receptor with Xenopus laevis erythrocytes establishes a substantial (2- to 10-fold) stimulation of the hybrid adenylate cyclase by the beta-agonist isoproterenol. The extent of stimulation depends on the amount of reconstituted beta-adrenergic receptor added, is blocked by propranolol, and is eliminated by boiling the beta-adrenergic receptor prior to reconstitution. The successful coupling of a purified receptor to a heterologous adenylate cyclase opens the way to the study of receptor structure---function relationships.

Functional integrity of desensitized beta-adrenergic receptors.

The adenylate cyclase-coupled beta 2-adrenergic receptor of the frog erythrocyte has served as a useful model system for elucidating the mechanisms of catecholamine-induced densensitization. In this system, it has been previously demonstrated that agonist-induced refractoriness is associated with sequestration of the beta-adrenergic receptors in vesicles away from the cell surface and from their effector unit, the adenylate cyclase system (Stadel, J.M., Strulovici, B., Nambi, P., Lavin, T.N., Briggs, M.M., Caron, M.G., and Lefkowitz, R.J. (1983) J. Biol. Chem. 258, 3032-3038). These internalized beta-adrenergic receptors appear to be structurally intact as assessed by photoaffinity labeling, but their functional status has previously been unknown. In the present studies, we sought to assess the functionality of the sequestered vesicular receptors by fusing them to Xenopus laevis erythrocytes. This cell is suitable for such studies, since it has almost no detectable beta-adrenergic receptor or catecholamine-sensitive adenylate cyclase, but contains prostaglandin E1-stimulable adenylate cyclase. Fusion of beta-adrenergic receptor-containing vesicles from desensitized frog erythrocytes with X. laevis erythrocytes results in a 30-fold stimulation of the hybrid adenylate cyclase by the beta-adrenergic agonist isoproterenol. This effect was entirely blocked by the beta-antagonist propranolol. The catecholamine-sensitive adenylate cyclase activity established in the vesicle-Xenopus hybrids showed the characteristic agonist potency series of the donor frog erythrocyte beta 2-adrenergic receptor. Fusion of vesicles from desensitized frog erythrocytes in which the beta-adrenergic receptors had been inactivated with the group specific reagent dicyclohexylcarbodiimide, or of vesicles derived from control frog erythrocytes, which contain low amounts of beta-adrenergic receptor, did not establish catecholamine-sensitive adenylate cyclase activity in the hybrids. These data demonstrate that beta-adrenergic receptors internalized during desensitization retain their functionality when recoupled to an adenylate cyclase system from a different source. The functional uncoupling of these receptors during desensitization is thus more likely due to their sequestration away from the other components of the adenylate cyclase than to any alterations in the receptors themselves.

Antibodies to the beta-adrenergic receptor: attenuation of catecholamine-sensitive adenylate cyclase and demonstration of postsynaptic receptor localization in brain.

Antibodies to the beta 2-adrenergic receptor of frog erythrocytes have been raised in rabbits by immunization with purified receptor preparations. Binding of the antibodies to the receptors was demonstrated by immunoprecipitation and by the altered mobility of the antibody-bound receptors on steric-exclusion HPLC columns. As assessed by a radioimmunoassay developed with the antibody, beta 2-adrenergic receptors from several sources showed various degrees of immunological crossreactivity whereas several beta 1-adrenergic receptors did not crossreact. The antibody appeared to not bind at the ligand binding site of the receptor and did not perturb antagonist radioligand binding to the receptor. Nonetheless, the antibodies selectively attenuated catecholamine-stimulated adenylate cyclase. This suggests that the antibodies recognize and bind to domains of the receptor other than the binding site and that may be involved in coupling to other components of the adenylate cyclase system. Immunocytochemical techniques were used with the antibodies to delineate a postsynaptic localization of beta-adrenergic receptors in rat and frog brain. Thus, these anti-beta-adrenergic receptor antibodies provide a useful reagent for probing beta-adrenergic receptor structure, function, and localization.

Desensitization of the beta-adrenergic receptor of frog erythrocytes. Recovery and characterization of the down-regulated receptors in sequestered vesicles.

Desensitization of the beta-adrenergic receptor of frog erythrocytes. Recovery and characterization of the down-regulated receptors in sequestered vesicles.

A study of mechanisms of post-irradiation changes in adenylate-cyclase activity.

Hormone-stimulated adenylate-cyclase activity (via beta-adrenergic receptors) is reduced after in vitro irradiation of plasma membranes of frog erythrocytes, at a dose (650 Gy) which does not change the number of beta-adrenergic receptors but causes an oxidative degradation of membrane lipids. Peroxidation of membrane lipids without irradiation also results in a lesser stimulation of adenylate cyclase by the beta-agonist. A possible mechanism of change in adenylate-cyclase functioning is discussed.

Re-evaluation of Feulgen stain for image cytometry: Part II. Application to electrochemically induced chromatin reactivation

Automated cytometry has been proposed by us as an extremely sensitive probe of the electrochemical reactivation of quiescent cells, provided that quantitative and specific staining techniques are available for absorption or fluorescence measurements. A complete re-evaluation is given of cell staining by means of the Feulgen reaction for absorption image cytometry, in order to obtain quantitative specificity for DNA amount and chromatin conformation. The possibility of computing from image cytometry the number of chromatin sites accessible to intercalation of small molecules, which should be related to template activity, was previously anticipated by us on an experimental basis, and is now assessed on a theoretical basis, linking together fluorescence flow cytometry and absorption image cytometry. The Feulgen staining technique has been applied to measure the amount of DNA during reactivation of frog erythrocytes and human lymphocytes induced by exposure to electromagnetic (e.m.) fields and by chemical changes of cell environment. Static automated absorption image analysis of Feulgen stained nuclei allows us to evaluate the onset of DNA synthesis and the effectiveness of the electrochemical stimulus on chromatin conformation.

532—Re-evaluation of Feulgen stain for image cytometry: Part II. Application to electrochemically induced chromatin reactivation

Automated cytometry has been proposed by us as an extremely sensitive probe of the electrochemical reactivation of quiescent cells, provided that quantitative and specific staining techniques are available for absorption or fluorescence measurements. A complete re-evaluation is given of cell staining by means of the Feulgen reaction for absorption image cytometry, in order to obtain quantitative specificity for DNA amount and chromatin conformation. The possibility of computing from image cytometry the number of chromatin sites accessible to intercalation of small molecules, which should be related to template activity, was previously anticipated by us on an experimental basis, and is now assessed on a theoretical basis, linking together fluorescence flow cytometry and absorption image cytometry. The Feulgen staining technique has been applied to measure the amount of DNA during reactivation of frog erythrocytes and human lymphocytes induced by exposure to electromagnetic (e.m.) fields and by chemical changes of cell environment. Static automated absorption image analysis of Feulgen stained nuclei allows us to evaluate the onset of DNA synthesis and the effectiveness of the electrochemical stimulus on chromatin conformation.

Molecular characterization of the β-adrenergic receptor of frog erythrocytes

The beta-adrenergic receptor of the frog erythrocyte has been solubilized in an active form with digitonin and purified by affinity chromatography and high performance liquid chromatography. Purified preparations contain a single band of iodinated protein of apparent M r = 58,000. This peptide appears to represent the ligand binding subunit of the receptor since purified preparations bind ligands with the same beta-adrenergic specificity as the solubilized or membrane-bound receptor, display the same isoelectric point and similar sedimentation characteristics in sucrose density gradients. The same ligand binding subunit can also be identified in partially purified receptor preparations or in membranes by photoaffinity labelling or photodependent crosslinking of two radiolabelled beta-adrenergic antagonists, p-azidobenzylcarazolol and p-aminobenzylcarazolol.

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.

Functional exchange of components between light-activated photoreceptor phosphodiesterase and hormone-activated adenylate cyclase systems.

Previous studies have noted profound similarities between the regulation of light-activated 3',5'-cyclic nucleotide phosphodiesterase (3',5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) in retinal rods and hormone-activated adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in a variety of tissues. We report here the functional exchange of components isolated from the photoreceptor system, which displayed predicted functional characteristics when incubated with recipient adenylate cyclase systems from rat cerebral cortical and hypothalamic synaptic membranes and frog erythrocyte ghosts. We demonstrate functional exchange of photoreceptor components at each of three loci: the hormone receptor, the GTP-binding protein (GBP), and the catalytic moiety of adenylate cyclase. Illuminated (but not unilluminated) rhodopsin was fund to mimic the hormone-receptor complex, causing GTP-dependent activation of adenylate cyclase. The photoreceptor GBP complexed with guanosine 5'-[beta, gamma)imidotriphosphate (p[NH]ppG) produced a marked activation of recipient adenylate cyclase systems. Much smaller activation was observed when GBP was not complexed with p[NH]ppG. A heat-stable photoreceptor phosphodiesterase inhibitor reduced both basal and Mn2+-activated adenylate cyclase activities and this inhibition was reversed by photoreceptor GBP.p[NH]ppG. These data demonstrate a remarkable functional compatibility between subunits of both systems and furthermore imply that specialized peptide domains responsible for protein-protein interactions are highly conserved.

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 β-adrenergic receptors: Identification of the β-receptor binding site(s) from turkey, pigeon, and frog erythrocyte

The β-adrenergic receptors in the erythrocyte membranes from turkey, pigeon, and frog have been identified in situ utilizing the photoaffinity label ±[ 125I]-iodoazidobenzylpindolol, ±[ 125I]IABP. The molecular weights determined by SDS-polyacrylamide gel electrophoresis are the following: turkey, 43,500; pigeon, 53,500, 46,000, and 45,000 [labeled in a ratio of 5 (53,500):2 (46,000 plus 45,000)]; and frog, a broad 60,000 to 67,000 dalton band. The data identify the binding site subunit(s) of these β-adrenergic receptors and suggest that the receptor structure from different β-receptor subtypes and different sources may be different. These biochemical differences may contribute to the pharmacologically observed distinction of β-receptor subtypes.