Biomedical subjects

C J Homcy

Publications and source records attributed to C J Homcy.

At least 73 records · Page 4Linked to original sources

PubMed1988

Impaired cardiac muscarinic receptor function in dogs with heart failure.

Prior physiological studies have suggested that parasympathetic control is altered in heart failure. The goal of our studies was to investigate the influence of heart failure on the muscarinic receptor, and its coupling to adenylate cyclase. Ligand binding studies using [3H]quinuclidinyl benzilate and enriched left ventricular (LV) sarcolemma, demonstrated that muscarinic receptor density in heart failure declined 36% from a control of 5.6 +/- 0.6 pmol/mg, with no change in antagonist affinity. However, agonist competition studies with both carbachol and oxotremorine showed that it was a loss of high affinity agonist binding sites in the sarcolemma from failing LV that accounted for this difference. The functional efficacy of the muscarinic receptor was also examined. When 1 microM methacholine was added to 0.1 mM GTP and 0.1 mM isoproterenol, adenylate cyclase stimulated activity was inhibited by 15% in normal LV but only 5% in LV sarcolemma from animals with heart failure even when the reduced adenylate cyclase in these heart failure animals was taken into account. Even at 100-fold greater concentrations of methacholine, significantly less inhibition of adenylate cyclase activity was observed in LV failure as compared with normal LV sarcolemma. Levels of the GTP-inhibitory protein known to couple the muscarinic receptor to adenylate cyclase, as measured with pertussis toxin labeling, were not depressed in LV failure. Thus, the inhibitory pathway regulating LV adenylate cyclase activity is defective in heart failure. The decrease in muscarinic receptor density, and in particular the specific loss of the high affinity agonist binding component of this receptor population, appears to be the major factor underlying this abnormality.

Adenylyl Cyclases↗

PubMed1988

Cellular processing of pro-atrial natriuretic factor (pro-ANF): studies using an antiserum that selectively binds ANF-(99-126) after its cleavage from pro-ANF.

Atrial natriuretic factor (ANF) is stored in atrial myocytes as a 15-17K prohormone, but circulates in plasma as a 3K, carboxy (C)-terminal fragment of the prohormone. The tissue location at which the cleavage of pro-ANF to its hormonal form occurs is unknown. In the present study, an immunological approach was taken to address this question. A polyclonal antiserum was generated which recognizes the hormonal form of ANF [ANF-(99-126)] only after its cleavage from the prohormone. This was accomplished by immunizing rabbits with a synthetic peptide corresponding to the seven amino (N)-terminal residues of ANF-(99-126) coupled to carrier protein via a C-terminal cysteine. This antiserum, anti-ANF-(99-105), demonstrated high affinity for ANF-(99-126) (IC50 = 170 pM), but displayed 100-fold less affinity for recombinant pro-ANF [ANF-(2-126)]. The N-terminal specificity of anti-ANF-(99-105) was evident by its failure to bind ANF-(103-126) at concentrations up to 100 nM. The specificity of anti-ANF-(99-105) for the hormonal form of ANF was examined by using thrombin to cleave pro-ANF and testing for the generation of anti-ANF-(99-105) immunoreactivity. Cleavage of atrial pro-ANF or 35S biosynthetically-labeled pro-ANF resulted in the production of immunoreactive material from the prohormone, whereas pro-ANF itself demonstrated no cross-reactivity with anti-ANF-(99-105). Anti-ANF-(99-105) could also recognize ANF released from the isolated perfused rat heart. When anti-ANF-(99-105) was used in immunohistochemical studies of rat atrial myocardium, no staining was observed in unfixed frozen sections. This suggests that proteolytic processing of pro-ANF is not an intracardiocytic event.

Amino Acid Sequence↗

PubMed1988

Electron microscopic localization of the beta-adrenergic receptor using a ferritin-alprenolol probe.

This report describes electron microscopic localization of the beta-adrenergic receptor using a beta-adrenergic receptor antagonist conjugated to ferritin. The conjugate was synthesized by reacting a carboxylic acid derivative of alprenolol with ferritin. The ferritin-alprenolol compound was shown to be effective in displacing specific [3H]dihydroalprenolol binding from rat erythrocyte membranes with a dissociation constant (Kd) of 25 nM. Rat erythrocyte ghosts were incubated with the compound and quantitative electron micrographic analysis yielded total binding of 1367 +/- 129 ferritin particles and nonspecific binding of 688 +/- 111 (six experiments). Thus, specific binding was 680 +/- 60 ferritin particles per red cell profile. Qualitative observations suggested that the particles were distributed randomly on the surface of the erythrocyte, although an occasional cluster was seen. A compound from another synthesis was shown be to effective in displacing specific [125I]iodocyanopindolol binding from neonatal rat cardiac myocyte membranes, with a dissociation constant of 13.8 nM, whereas native alprenolol had a dissociation constant of 1.3 nM. Neonatal rat cardiac myocytes were incubated with the compound and processed for electron microscopy. Total binding along the sarcolemmal membrane was 504 +/- 38 ferritin particles/100 micron of membrane and nonspecific binding was 301 +/- 26 ferritin particles/100 micron of membrane (seven experiments), yielding specific binding of 203 ferritin particles/100 micron of membrane. In additional studies, specific binding was inhibited 95% with 10(-5) M l-isoproterenol and 29% with d-isoproterenol, indicating stereoselectivity (seven experiments). The probe was distributed randomly along the sarcolemma with no preferential localization to coated pits or other membrane specializations. From measurements of the surface area of the average cardiac myocyte (732 micron 2), the specific binding of ferritin-alprenolol per 100 micron of membrane (203), and section thickness (0.08 micron), we calculated that cardiac myocytes had 18,575 beta-adrenergic membrane receptor sites. Thus, we have described a method for synthesizing and applying an electron-dense probe for electron microscopic localization of beta-adrenergic receptors. In these studies we determined the distribution of these receptors on rat erythrocyte ghosts and neonatal rat cardiac myocytes.

Alprenolol↗

PubMed1987

Characterization and purification of a protease in serum that cleaves proatrial natriuretic factor (ProANF) to its circulating forms.

Atrial natriuretic factor (ANF) is synthesized and stored in atrial cardiocytes as a 17-kilodalton (kDa), 126 amino acid polypeptide, proANF, but circulates as smaller, 24 and 28 amino acid peptide fragments of the carboxy terminus of proANF. It has previously been shown that proANF is secreted intact from cultured atrial cardiocytes and can be cleaved by a serum protease to smaller, 3-kDa peptides believed to be the circulating forms. This report describes the purification and characterization of this proANF-cleaving protease from rat serum. The cleavages both of 35S-labeled proANF derived from rat atrial cell cultures, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/autoradiography, and of a synthetic p-nitroanilide-containing substrate were used as assays for the detection of enzyme activity. ProANF-cleaving activity was found in rat serum, with no such activity detectable in rat plasma. Cleavage in serum was not dependent on the presence of platelets or other cellular elements. Complete inhibition of proANF cleavage was obtained with the protease inhibitors benzamidine, leupeptin, phenylmethanesulfonyl fluoride, and diisopropyl fluorophosphate (DFP) but not with aprotinin, soybean trypsin inhibitor, pepstatin, or hirudin. Unlike the vitamin K dependent plasma proteins, the proANF-cleaving protease did not adsorb to barium sulfate. With the sequential application of ion-exchange, hydroxylapatite, lectin affinity, and gel filtration chromatography, a 5000-6000-fold purification of the enzyme from rat serum was achieved. Fractionation of either whole serum or the purified enzyme by gel filtration chromatography revealed a single peak of activity corresponding to a protein with a Stokes radius of 45 A.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Analysis of atrial natriuretic factor biosynthesis and secretion in adult and neonatal rat atrial cardiocytes.

Atrial natriuretic factor (ANF) is stored in atrial cardiocytes as the 126 amino acid polypeptide, proANF, which is later cleaved to the 24-28 amino acid carboxyterminal peptides, the major circulating forms. Earlier studies have demonstrated that isolated, cultured neonatal rat cardiocytes both store and secrete proANF, which can be cleaved to the smaller circulating form(s) by a serum protease. Since differences may exist between neonatal and adult cardiocytes with respect to ANF synthesis and processing, we compared the forms of ANF stored and secreted by neonatal rat cardiocytes with those of adult cells. Using four to five day cultures of isolated atrial cardiocytes prepared from the hearts of neonatal and adult rats, pulse-chase studies were performed with 35S-cysteine and 35S-methionine. Analysis of ANF stored and secreted by these cells was performed by immunoprecipitation of cell extracts and culture media using antibodies directed to either the carboxyterminus or aminoterminus of proANF followed by SDS-PAGE and autoradiography. Cell extracts from both adult and neonatal cultures were found to contain only a 17-kDa polypeptide, previously identified as proANF. The predominant form found in the culture media was also the 17-kDa peptide, with smaller quantities of its 3-kDa carboxyterminal and 14-kDa aminoterminal cleavage products. We conclude from these studies that proANF is the major form stored and secreted by both adult and neonatal cardiocytes in culture; the activity of the protease that cleaves proANF to the smaller forms found in the circulation is either attenuated or is overwhelmed by high ANF-secretory rates in these cultures. Alternatively, the ANF processing and secretory pathways may be somehow altered in culture such that proANF escapes protease cleavage. Further studies will elucidate the nature and location of this protease.

Animals↗

PubMed1987

Enhanced antigen-antibody binding affinity mediated by an anti-idiotypic antibody.

We previously described the production of four monoclonal antibodies to the beta-adrenergic receptor antagonist alprenolol. One of these antibodies, 5B7 (IgG2a, kappa), was used to raise anti-idiotypic antisera in rabbits. In contrast to the expected results, one of the anti-idiotypic antisera (R9) promotes [125I]iodocyanopindolol (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 R9 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 [k-1(+R9) = 0.025 min-1 vs. k-1(-R9) = 2.04 min-1], consistent with the 100-fold change 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Alprenolol↗

PubMed1987

Free radical-mediated membrane depolarization in renal and cardiac cells.

Cell membrane potential was measured with a flow cytometer by quantitating the intracellular accumulation of a fluorescent cationic carbocyanine dye. We used this system to demonstrate depolarization upon the addition of hydrogen peroxide (10-1,000 microM) and ferrous chloride (25-100 microM) to cultures of either neonatal rat myocardial or LLC-PK1 renal epithelial cells. Ferrous chloride-induced depolarization was prevented by superoxide dismutase, catalase and dimethyl sulfoxide, suggesting roles for the superoxide anion, hydrogen peroxide and the hydroxyl radical in effecting this depolarization, possibly through a Fenton-type reaction mechanism. Supplementation of either cell type with 2 microM tocopherol acid succinate during growth in tissue culture, prior to exposure to the oxidizing agent, decreased the magnitude of the depolarization in both cell types. The results are consistent with a role for tocopherols in scavenging free radical species responsible for the depolarization of the cell membrane.

Animals↗

PubMed1987

Oxygen radicals alter the cell membrane potential in a renal cell line (LLC-PK1) with differentiated characteristics of proximal tubular cells.

We employed a carbocyanine dye (1,1',3,3,3',3'-hexamethylindocarbocyanine iodide) to measure the plasma membrane potential of LLC-PK1 renal epithelial cells exposed to either xanthine oxidase-generated oxygen radicals or to hydrogen peroxide. Measurements were performed using a fluorescent-activated cell sorter to record fluorescence on a cell by cell basis. Initial exposure of cells to low concentrations of either H2O2 or xanthine oxidase resulted in a transient increase in membrane potential relative to control cells (P less than 0.001), followed by an exponential decline in potential (P less than 0.001). The addition of extracellular catalase diminished the H2O2-related decline in potential, consistent with a role for hydrogen peroxide in producing this effect. Pretreatment of cells with inhibitors of intracellular catalase and superoxide dismutase prior to exposure to xanthine oxidase caused an even larger decline in potential (P less than 0.001). Cells could be partially protected from the radical-mediated loss of potential by incubating them in a hypertonic (400 mosmolal) environment during radical exposure. Similarly, the loss of membrane potential was increased after incubation of cells in a hypotonic (200 mosmolal) environment during radical exposure. These observations are consistent with a reduction in membrane potential effected by exposure to oxygen radicals (including superoxide anion and hydrogen peroxide). This reduction may be prevented, in part, by radical scavenging enzymes and by reducing the degree of cellular swelling in response to oxygen radical exposure.

Carbocyanines↗

PubMed1987

High affinity binding of reovirus type 3 to cells that lack beta adrenergic receptor activity.

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.

Adenylyl Cyclases↗

PubMed1987

Evidence that the sigma 1 protein of reovirus serotype 3 is a multimer.

In this report, we study the reovirus serotype 3 (strain Dearing) sigma 1 protein obtained from various sources: from Escherichia coli expressing sigma 1 protein, from reovirus-infected mouse L cells, and from purified reovirions. We demonstrate that the sigma 1 protein is a multimer in its undisrupted form and present biochemical evidence suggesting that the multimer is made up of four sigma 1 subunits.

Animals↗

PubMed1987

A serum protease cleaves proANF into a 14-kilodalton peptide and ANF.

Proatrial natriuretic factor (proANF), the 126-amino acid precursor of ANF, is the major storage form in mammalian atria. In contrast, two ANF peptides containing the 28- and 24-carboxyterminal residues of proANF have been isolated from rat plasma. Whether the cleavage of proANF in vivo to these ANF peptides occurs during or after its release into the circulation has not been determined. The latter possibility was suggested by our previous study where, by using a cultured rat cardiocyte preparation, we demonstrated that proANF is secreted intact into the culture medium. We now report that serum, but not plasma, contains a protease that specifically cleaves the 17-kdalton proANF to a 14-kdalton amino-terminal peptide and the carboxyterminal 3-kdalton circulating forms of ANF. The role of this proANF-cleaving enzyme in the generation of the biologically active ANF peptides remains to be defined. Its isolation and characterization should provide insights into its site of production and whether in vivo it is involved in the processing of circulating proANF.

Animals↗

PubMed1987

Synthesis and characterization of arylamine derivatives of rauwolscine as molecular probes for alpha 2-adrenergic receptors.

The selective alpha 2-adrenergic receptor antagonist rauwolscine was structurally modified to yield a series of arylamine carboxamide derivatives, which were investigated as potential molecular probes for the localization and structural characterization of alpha 2-adrenergic receptors. The arylamine carboxamides differ in the number of carbon atoms separating the reactive phenyl moiety from the fused ring structure of the parent compound, rauwolscine carboxylate. Competitive inhibition studies with [3H]rauwolscine in rat kidney membranes indicate that the affinity for the carboxamide derivatives is inversely related to the length of the carbon spacer arm with rauwolscine 4-aminophenyl carboxamide (zero carbon spacer arm; rau-AMPC) exhibiting the highest affinity (Kd = 2.3 +/- 0.2 nM). Radioiodination of rau-AMPC yields a ligand, 125I-rau-AMPC, which binds to rat kidney alpha 2-adrenergic receptors with high affinity, as determined by both kinetic analysis (Kd = k2/k1 = 0.016 min-1/2.1 X 10(7) M-1 min-1 = 0.76 nM) and equilibrium binding studies (Kd = 0.78 +/- 0.16 nM). 125I-rau-AMPC was quantitatively converted to the photolabile arylazide derivative 17 alpha-hydroxy-20 alpha-yohimban-16 beta-(N-4-azido-3-[125I]iodophenyl) carboxamide (125I-rau-AZPC). In a partially purified receptor preparation from porcine brain, this compound photolabels a major (Mr = 62,000) peptide. The labeling of this peptide is inhibited by adrenergic agonists and antagonists with a rank order of potency consistent with an alpha 2-adrenergic receptor binding site. Both 125I-rau-AMPC and the photolabile arylazide derivative, 125I-rau-AZPC, should prove useful as molecular probes for the structural and biochemical characterization of alpha 2-adrenergic receptors.

Adrenergic beta-Agonists↗

PubMed1987

Structural properties of the alpha 1-adrenergic receptor: studies with membrane and purified receptor preparations.

The contribution of hydrophobic, ionic, and disulfide bonds in the alpha 1-adrenergic receptor structure was evaluated using rat hepatic plasma membrane and purified receptor preparations. In addition, the findings were compared with similar structural evaluations of the beta 2-adrenergic receptor purified from canine lung. Both the alpha 1- and beta 2-adrenergic receptors contain a disulfide bond critical for ligand binding. However, the disulfide bond in the beta-receptor structure is accessible to solvent, while the disulfide bond within the alpha-receptor structure is "masked" and inaccessible to solvent. Reduction of this disulfide bond requires initial denaturation of the alpha 1-receptor. Although the tertiary structures of both receptor proteins appear to be stabilized by strong hydrophobic interactions, differential sensitivities are observed with a variety of structural perturbants, including high concentrations of sodium chloride, guanidine hydrochloride, and ethylene glycol.

Animals↗

PubMed1986

Agonist interactions at hepatic alpha 1- and beta-adrenergic receptors: affinity-state regulation by guanine nucleotides and temperature.

We investigated the binding characteristics of agonists to alpha 1- and beta-adrenergic receptors of intact liver cells, broken rat liver cell membranes, and detergent-solubilized preparations under varying experimental conditions, focusing on the different "states" of the receptor for agonists and the regulation of these states by temperature and guanine nucleotides. While only low-affinity binding of agonists to both receptor subtypes was evident in studies performed at 37 degrees C with solubilized preparations, biphasic competition curves for agonists were observed in both intact cells and membrane preparations; the majority of sites were of low affinity. In membrane preparations, the nonhydrolyzable GTP analogue Gpp(NH)p caused a rightward shift of agonist competition curves and a loss of high-affinity binding. These results are consistent with the involvement of guanine nucleotide binding proteins in both alpha 1- and beta-adrenergic transduction pathways. When competition studies were performed at 4 degrees C, receptor sites existed predominantly in the high-affinity configuration, in intact cells and membranes, as well as in soluble preparations. In contrast to the studies conducted at 37 degrees C, no Gpp(NH)p-induced conversion to the lower affinity state could be demonstrated in studies performed with membrane preparations at 4 degrees C. Thus, the high-affinity state of alpha 1- and beta-adrenergic receptors is stabilized at 4 degrees C in intact cells, membranes, and soluble preparations. After incubations had been performed at 37 degrees C, high-affinity binding of agonists could not be restored by subsequent incubation at 4 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic alpha-Agonists↗

PubMed1986

Serum-mediated enhancement of ANF accumulation in the culture medium of cardiac atriocytes.

Cultured cardiac myocytes provide a useful system for investigating ANF biosynthesis and regulation. It has previously been demonstrated that the predominant form of ANF stored in and released from this myocyte model is the 17 kD prohormone, proANF. We report here that as quantitated both by radioimmunoassay and by SDS-PAGE of intrinsically labelled ANF released from these cardiocytes, the addition of serum promotes a 5-6 fold increase in ANF accumulation in the medium of these cells as compared to ANF accumulation in the presence of a defined chemical medium alone. The stimulating effect of serum is immediate and persists in a linear manner for at least 120 minutes. This effect of serum can be reproduced by the addition of albumin or other proteins to the medium but not by alterations in osmolality. Whether this phenomenon represents enhanced release of proANF or is secondary to inhibition of proANF degradation has yet to be determined.

Atrial Natriuretic Factor↗

PubMed1986

Purification and characterization of the beta 2-adrenergic receptor from calf lung.

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.

Animals↗

PubMed1986

NADP improves the efficiency of cholera toxin catalyzed ADP-ribosylation in liver and heart membranes.

Guanine nucleotide binding proteins (G-proteins) can be identified by their ability to be ADP-ribosylated using [32P]NAD as the substrate and bacterial toxins as catalysts. This labelling, when performed in liver and sarcolemma membrane preparations, can be complicated by competing enzymes which degrade NAD, making it unavailable to participate in the desired reaction. The addition of NADP in reaction mixtures markedly slows the degradation of NAD, but does not compete with NAD in cholera toxin labelling of stimulatory G-protein. The efficiency of cholera toxin labelling is improved to the extent that saturation curves may be constructed, allowing the quantitation of ADP-ribosylation sites in membranes.

Animals↗

PubMed1986

Agonist-induced isomerization of the alpha 1-adrenergic receptor: kinetic analysis using broken-cell and solubilized preparations.

The affinity of agonists but not antagonists at hepatic membrane alpha 1-adrenergic receptors is temperature dependent; a 100-fold higher affinity is observed at 4 degrees C than at 37 degrees C. The relationship between these two agonist affinity states was investigated by using a strategy that allows the kinetics of this transition to be examined under equilibrium conditions. When competition assays are performed at 37 degrees C for varying intervals and the reaction mixture is then rapidly cooled by freezing, allowed to thaw, and further equilibrated at 4 degrees C, a rapid and progressive decrease (t1/2 of 1-2 min) in agonist affinity occurs, the extent of which is directly related to the incubation time at 37 degrees C. This decrease in agonist affinity is sustained as long as agonist is present but can be reversed by its subsequent removal. In contrast, no change in affinity is seen in identical experiments when antagonists are employed as the competing ligand. High-affinity binding of agonists is also demonstrated in short-term nonequilibrium experiments, indicating that the low-temperature incubations do not induce, but rather stabilize, a receptor conformation of high affinity for agonists. These findings suggest that the predominantly low-affinity binding of agonists to alpha 1-adrenergic receptors demonstrated in equilibrium studies at physiological temperatures may be the result of a ligand-driven decrease in affinity. Since the transition in receptor affinity for agonists occurs not only in broken-cell preparations but also after detergent solubilization of the membrane receptor, it most likely is due to an agonist-induced change in the conformation of the receptor protein per se.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗