Isoproterenol stimulates lipid methylation in C6 cells without affecting membrane fluidity

C6 glioma cells were stimulated with the beta-adrenergic agonist isoproterenol. The rate of cyclic AMP accumulation and lipid methylation increased. However, the elevated rate of lipid methylation did not affect the fluidity of the plasma membrane. It is concluded that lipid methylation is not responsible for making the plasma membrane more fluid during the beta-adrenergic response.     

Control of salivary phospholipid content and composition.

The mediation of phospholipid secretion in rat sublingual salivary gland cells maintained in the presence of [3H]choline was investigated. The secretion of [3H]choline-containing phospholipids was enhanced by beta-adrenergic agonist, isoproterenol, to a greater extent than the cholinergic agonist carbachol. A 2.9-fold increase in phospholipid secretion occurred with isoproterenol, while carbachol evoked only about 1.3-fold increase. In contrast to carbachol, the enhanced phospholipid secretion due to isoproterenol was accompanied by an increase in cAMP concentration. The secretion of phospholipids was also stimulated by dibutyryl-cAMP and the protein kinase C activator, phorbol myristate acetate, but not by 4 alpha-phorbol 12, 13-didecanoate which does not activate protein kinase C. Furthermore, the effects of dibutyryl-cAMP and phorbol myristate acetate were additive. The phospholipids secreted in response to isoproterenol exhibited a 52% decrease in lysophosphatidylcholine, while those secreted in response to carbachol showed a 23% lower content of phosphatidylcholine, and were enriched in lysophosphatidylcholine (2.8-fold) and sphingomyelin (1.4-fold). The results suggest that salivary phospholipid secretion remains mainly under beta-adrenergic control, while the phospholipid makeup is under cholinergic regulation.     

Secretion of gastric mucus phospholipids in response to beta-adrenergic G protein-coupled receptor activation is mediated by SRC kinase-dependent epidermal growth factor receptor transactivation.

Recent advances in understanding the nature of cellular responses mediated by G protein-coupled receptor (GPCR) activation indicate that integration of the converging regulatory signals into functional cellular pathways requires epidermal growth factor receptor (EGFR) transactivation. In this study, we report that G protein-coupled beta-adrenergic receptor activation leading to stimulation in gastric mucus phospholipid secretion occurs with the involvement of EGFR. Using [14C]choline-labeled gastric mucosal cells in culture, we show that stimulatory effect of beta-adrenergic agonist, isoproterenol, on phospholipid release was subject to a dose-dependent suppression by EGFR kinase inhibitor, PD153035, as well as wortmannin, a specific inhibitor of PI3K. Both inhibitors, moreover, caused the reduction in the gastric mucosal cell phospholipid secretory responses to beta-adrenergic agonist-generated second messenger, cAMP as well as adenyl cyclase activator, forskolin. The gastric mucosal phospholipid secretory responses to isoproterenol, furthermore, were inhibited by PP2, a selective inhibitor of tyrosine kinase Src responsible for ligand-independent EGFR phosphorylation, but not by ERK inhibitor, PD98059. The inhibition of ERK, moreover, did not cause attenuation in phospholipid secretory responses to cAMP and forskolin. The findings underline the central role of EGFR in mediation of gastric mucosal secretory processes, and demonstrate the requirement for Src kinase-dependent EGFR transactivation in regulation of gastric mucosal phospholipid secretion in response to beta-adrenergic GPCR activation.     

Alteration of membrane phospholipid methylation by adenosine analogs does not affect T lymphocyte activation

Membrane phospholipid methylation has been described during activation of various immune cells. Moreover recent data indicated modulation of immune cells functions by adenosine. As S-Adenosyl-methionine and S-Adenosyl-homocysteine are adenosine analogs and modulators of transmethylation reactions, the effects of SAH and SAM were investigated on membrane phospholipid methylation and lymphocyte activation. SAM (10(-5) M) was shown to induce the membrane phospholipid methylation as assessed by the 3H-methyl-incorporation in membrane extract. This effect was inhibited by SAH. In contrast SAM and SAH did not affect the phytohemagglutinin-induced proliferative response of peripheral blood mononuclear cells. SAH neither modified the early internalization of membrane CD3 antigens nor did it prevent the late expression of HLA-DR antigens on lymphocytes activated by phytohemagglutinin. These results indicate that in vitro alteration of phospholipid methylation does not affect subsequent steps of human T lymphocyte activation and proliferation.     

Role of adrenergic and cholinergic mediators in salivary phospholipids secretion.

The influence of adrenergic and cholinergic mediators on phospholipid secretion in rat sublingual salivary gland cells maintained in the presence of [3H]choline was investigated. The secretion of [3H]choline-containing phospholipids over 30 min period averaged 1.93% of the total cellular labeled phospholipids in the absence of any mediator, and was enhanced by beta-adrenergic agonist, isoproterenol, to a greater extent than the cholinergic agonists, pilocarpine and carbachol. A 2.9-fold increase in phospholipid secretion occurred with isoproterenol, while pilocarpine and carbachol evoked only 1.3-fold increase. The effect of isoproterenol was inhibited by alprenolol and that of pilocarpine and carbachol by atropine. In contrast to pilocarpine and carbachol, the enhanced phospholipid secretion due to isoproterenol was accompanied by an increase in cAMP concentration. The secretion of phospholipids was also stimulated by dibutyryl-cAMP and the protein kinase C activator, phorbol myristate acetate, but not by 4 alpha-phorbol 12,13-didecanoate which does not activate protein kinase C. Furthermore, the effects of dibutyryl-cAMP and phorbol myristate acetate were additive. The phospholipids secreted in response to isoproterenol exhibited a 52% decrease in lysophosphatidylcholine, while those secreted in response to pilocarpine and carbachol showed a 21-23% lower content of phosphatidylcholine, and were enriched in lysophosphatidylcholine (2.6-2.8-fold) and sphingomyelin (1.5-1.6-fold). The results indicate that salivary phospholipid secretion remains mainly under beta-adrenergic regulation, while the phospholipid makeup of the secretion is under cholinergic control.     

Salivary phospholipid secretion in response to beta-adrenergic stimulation is mediated by Src kinase-dependent epidermal growth factor receptor transactivation

Communication between receptor tyrosine kinase and G protein-coupled receptor (GPCR)-mediated signaling is recognized as a common integrator linking diverse aspects of intracellular signaling systems. Here, we report that G protein-coupled beta-adrenergic receptor activation leading to stimulation of salivary phospholipid release occurs with the involvement of epidermal growth factor receptor (EGFR). Using sublingual gland acinar cells, we show that prosecretory effect of isoproterenol on phospholipid release was subjected to suppression by EGFR kinase inhibitor, PD153035, and wortmannin, an inhibitor of PI3K, but not by PD98059, an inhibitor of extracellular signal regulated kinase (ERK). Furthermore, wortmannin, but not the ERK inhibitor, caused the reduction in the acinar cell secretory responses to beta-adrenergic agonist-generated cAMP as well as adenyl cyclase activator, forskolin. The acinar cell phospholipid secretory responses to isoproterenol, moreover, were inhibited by PP2, a selective inhibitor of tyrosine kinase Src responsible for ligand-independent EGFR phosphorylation. Taken together, our data are the first to demonstrate the requirement for Src kinase-dependent EGFR transactivation in regulation of salivary phospholipid secretion in response to beta-adrenergic GPCR activation.     

Fatty acid uptake and catecholamine-stimulated phospholipid metabolism in immortalized airway epithelial cells established from primary cultures

The incorporation of [14C]oleic and [14C]linoleic acid into phospholipids and neutral lipids was compared in two recently immortalized airway epithelial cell lines. In addition, the effects of adrenergic stimulation on phospholipid turnover was examined. Both cell lines readily incorporated the fatty acids into all phospholipid and neutral lipid fractions. Isoproterenol (1 microM) induced Ca2+ transients in both cell lines, indicating a functional beta-adrenergic response. Epinephrine (10 microM; 15 min) stimulation of cells prelabeled with [14C]linoleic acid increased the percentage of label in phosphatidylcholine in one cell line. Lipid metabolism can now be extensively studied in human airway epithelia.     

Phospholipid Methylation Increases [3H]Diazepam and [3H]GABA Binding in Membrane Preparations of Rat Cerebellum

The effect of phospholipid methylation on both [3H]diazepam and [3H]GABA ( [3H]gamma-aminobutyric acid) binding to crude synaptic plasma membrane from rat cerebellum has been studied. S-Adenosylmethionine (SAM) stimulates [3H]methyl group incorporation into membrane phospholipids and enhances [3H]diazepam binding by increasing the apparent Bmax. Conversely, inhibition of [3H]methyl group transfer from [3H]SAM to phospholipids by preincubation with SAM at 0 degrees C or with SAH abolishes the increase of binding. After preincubation with SAM, analysis of the GABA binding reveals the presence of binding sites with high affinity, a property absent in control membranes preincubated without SAM. Among the neurotransmitter bindings tested, only those of GABA and benzodiazepine in the cerebellum and beta-adrenergic ligands in the cerebral cortex are enhanced upon stimulation of phospholipid methyltransferase activity. [3H]Dihydromorphine, [3H]dihydro-alpha-ergokryptine and [3H]spiroperidol bindings are not affected by SAM. The present data suggest an involvement of phospholipid methylation in regulation of both [3H]GABA and [3H]-diazepam binding.     

Differences in the beta-adrenergic responsiveness between high and low passage rat glioma C6 cells

Responsiveness to catecholamines was studied in two different strains of rat glioma C6 cells. The C6 cells of low passage possessed a high capacity to accumulate cyclic AMP in response to (-)-isoproterenol. Cholera toxin was also able to stimulate cyclic AMP accumulation in these cells. High passage C6 cells were unresponsive to (-)-isoproterenol or to cholera toxin except in the presence of a high concentration of phosphodiesterase inhibitor. The affinity of beta-adrenergic receptors on both strains for (-) [3H] dihydroalprenolol was similar; however, C6 low passage possessed several times the number of beta-adrenergic receptors found in C6 high passage. This difference correlated with the difference found in (-)-isoproterenol-stimulated adenylate cyclase between C6 low passage and high passage. The sodium fluoride-stimulated adenylate cyclase was similar in both strains. Cyclic AMP phosphodiesterase activity was 2-3 times higher in homogenates of C6 high passage than in low passage. In intact cells, the rate of breakdown of cyclic AMP was 5-times faster in C6 high passage than in low passage. Thus, differences in beta-adrenergic receptor number and phosphodiesterase activity explain in part the lack of responsiveness of C6 high passage. Our studies indicate that continuous subculturing of rat glioma C6 cells led to complex alterations in the beta-adrenergic receptor-adenylate cyclase system.     

Phospholipid methylation by intact rat Leydig cells

Phospholipid methylation by intact Leydig cells was investigated by determining the incorporation of radioactivity from [3H-methyl] methionine into phospholipids. Leydig cells incorporated significantly more radioactivity into phospholipids than did unpurified testicular cells, non-Leydig testicular cells, or red blood cells. Approximately 40% of the radioactivity was found in phosphatidylcholine, indicating that the methyltransferase pathway for the synthesis of this phospholipid is highly active in rat Leydig cells. Addition of luteinizing hormone to cells preloaded with [3H-methyl] methionine did not alter the rate of phospholipid methylation. However, phospholipid methylation by Leydig cells desensitized by the injection of human chorionic gonadotropin 1 to 7 days previously was reduced by approximately 60%. Inhibition of phospholipid methylation to 75% of normal with homocysteine thiolactone did not affect luteinizing hormone-stimulated androgen production. Further inhibition of phospholipid (and protein) methylation by treatment with homocysteine thiolactone and 3-deazaadenosine significantly reduced luteinizing hormone-stimulated androgen production. The results of this study demonstrate that the methyltransferase pathway for the synthesis of phosphatidylcholine is highly active in intact Leydig cells but is reduced in desensitized Leydig cells. There does not appear to be a close association between the activity of this pathway and the ability of luteinizing hormone to acutely stimulate androgen production.     

The interaction of dietary fatty acid and cholesterol on catecholamine-stimulated adenylate cyclase activity in the rat heart

Diets supplemented with high levels of saturated or unsaturated fatty acids supplied by addition of sheep kidney fat or sunflower seed oil, respectively, were fed to rats with or without dietary cholesterol. The effects of these diets on cardiac membrane lipid composition, catecholamine-stimulated adenylate cyclase and beta-adrenergic receptor activity associated with cardiac membranes, were determined. The fatty acid-supplemented diets, either with or without cholesterol, resulted in alterations in the proportion of the (n-6) to (n-3) series of unsaturated fatty acids, with the sunflower seed oil increasing and the sheep kidney fat decreasing this ratio, but did not by themselves significantly alter the ratio of saturated to unsaturated fatty acids. However, cholesterol supplementation resulted in a decrease in the proportion of saturated and polyunsaturated fatty acids and a dramatic increase in oleic acid in cardiac membrane phospholipids irrespective of the nature of the dietary fatty acid supplement. The cholesterol/phospholipid ratio of cardiac membrane lipids was also markedly increased with dietary cholesterol supplementation. Although relatively unaffected by the nature of the dietary fatty acid supplement, catecholamine-stimulated adenylate cyclase activity was significantly increased with dietary cholesterol supplementation and was positively correlated with the value of the membrane cholesterol/phospholipid ratio. Although the dissociation constant for the beta-adrenergic receptor, determined by [125I](-)-iodocyanopindolol binding, was unaffected by the nature of the dietary lipid supplement, the number of beta-adrenergic receptors was dramatically reduced by dietary cholesterol and negatively correlated with the value of the membrane cholesterol/phospholipid ratio. These results indicate that the activity of the membrane-associated beta-adrenergic/adenylate cyclase system of the heart can be influenced by dietary lipids particularly those altering the membrane cholesterol/phospholipid ratio and presumably membrane physico-chemical properties. In the face of these dietary-induced changes, a degree of homeostasis was apparent both with regard to membrane fatty acid composition in response to an altered membrane cholesterol/phospholipid ratio, and to down regulation of the beta-adrenergic receptor in response to enhanced catecholamine-stimulated adenylate cyclase activity.     

Plasma from uninephrectomized rats stimulates phospholipid methylation and arachidonic acid release in renal-cortical slices.

Phospholipid methylation and arachidonic acid release in renal-cortical slices was investigated in vitro after addition of plasma from uninephrectomized or sham-operated rats. Plasma from uninephrectomized rats ('uni-plasma') stimulated phospholipid methylation when obtained within the first 3 h after uninephrectomy. With different amounts of added plasma a graded response in phospholipid methylation was obtained. Addition of 50 nM-12-O-tetradecanoylphorbol 13-acetate for 10 min to intact slices also stimulated phospholipid methylation, whereas incubation of slices before addition of 'uni-plasma' with 100 microM-1-(5-isoquinolinylsulphonyl)-2-methylpiperazine prevented it, suggesting that protein kinase C stimulates phospholipid methylation in renal-cortical slices. Plasma from uninephrectomized rats also stimulates [3H]arachidonic acid release from phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) via activation of phospholipase A2. Two mechanisms of phospholipase A2 activation are proposed: first, in which it is activated by protein kinase C and releases 3H radioactivity from PtdCho, and second, in which phospholipase A2 is stimulated by Ca2+ ions and releases 3H radioactivity from PtdEtn.     

Calcium signals and phospholipid methylation in eukaryotic cells.

Rat basophil leukaemic (2H3) cells, mast cells and mouse thymocytes respond to stimulation by specific ligands with an increase in the free cytosolic Ca2+ concentration. The time courses of these Ca signals and the biological responses have been compared with changes in phospholipid metabolism. Increased phosphoinositide metabolism coincides with the Ca signals and the responses in each cell system, whereas any increase in phospholipid methylation during the response is less than one molecule per receptor and at least 5-50-fold less than the increases reported previously. Furthermore, no significant changes were detected in the concentration of S-adenosylmethionine, the methyl-group donor in the synthesis of methylated phospholipids. The hypothesis that phospholipid methylation is obligatory for receptor-mediated Ca signals is not supported by these data and requires critical re-evaluation.     

Reduction in beta-adrenergic response of cultured glioma cells following depletion of intracellular GTP.

1. When C6 glioma cells were incubated with mycophenolic acid, a potent and specific inhibitor of IMP:NAD oxidoreductase (EC 1.2.1.14) there was a marked depletion of the cellular content of GTP. The viability of the cells was unaffected. 2. The adenosine 3':5'-monophosphate (cyclic AMP) response of C6 glioma cells to the beta-adrenergic stimulant, (+/-)isoprenaline, was considerably reduced after treatment with mycophenolic acid. The diminished response to (+/-)isoprenaline was prevented by the inclusion of guanine in the culture medium along with mycophenolic acid. 3. The adenylate cyclase response to (+/-)isoprenaline of whole homogenates from C6 cells treated with mycophenolic acid was also depressed; the response was restored to normal by the addition of GTP. 4. The adenylate cyclase response to (+/-)isoprenaline of a membrane fraction prepared from homogenates of C6 cells was almost totally dependent on the presence of added GTP. Membrane fractions from control and mycophenolic-acid-treated C6 cells gave similar adenylate cyclase responses to (+/-)isoprenaline in the presence of GTP. 5. It is concluded that mycophenolic acid may depress the beta-adrenergic sensitivity of C6 cells by depleting the cellular content of GTP.     

Does Phospholipid Methylation Play a Role in the Primary Mechanism of Action of Nerve Growth Factor?

Nerve growth factor (NGF)-untreated (naive) and neurite-bearing NGF-treated ("primed") PC12 rat pheochromocytoma cells were used as model system to study the role of phospholipid methylation in the NGF mechanism of action. The neurite-bearing cultures were deprived of NGF for 3 h before experimentation. Under both experimental conditions, the cells were labelled with [methyl-3H]methionine and then challenged with NGF for time periods ranging from 5 s to 30 min. Methylated phospholipids were extracted and then resolved and identified by TLC as phosphatidyl mono-, di-, and trimethyl ethanolamine. Quantification of the amount of radioactivity incorporated into each of the phospholipids indicated that NGF does not significantly alter phospholipid methylation either in naive or in neurite-bearing cells. Furthermore, using a methyltransferase inhibitor, it was found that neurite outgrowth still occurs when phospholipid methylation is almost completely blocked. These results indicate that phospholipid methylation does not play a primary role in the mechanism of action of NGF.     

Serum catecholamines desensitize beta-adrenergic receptors of cultured C6 glioma cells

C6 glioma cells grown in medium containing fetal bovine serum have a decreased beta-adrenergic receptor number and beta-receptor-stimulated cyclic AMP accumulation as compared to cells grown in a serum-free, defined medium. The decreased number of receptors and decreased cAMP accumulation are attributable to a suppression of receptor binding and response by serum as opposed to increases produced by growth in the defined medium. Serum, when added to cells grown in the absence of serum, stimulated cellular cyclic AMP levels to 2-3 times basal levels. This direct stimulatory effect was blocked by incubation of the cells with the beta-adrenergic antagonist propranolol and was partially reversed by dialysis of the serum. In contrast, addition of serum to cells that have been grown with serum fails to stimulate cyclic AMP accumulation. The decrease in receptors following growth in serum can be mimicked by growing cells in serum-free medium in the presence of beta-adrenergic agonists such as isoproterenol or norepinephrine. Radioenzymatic assays indicate that fetal bovine serum contains approximately 0.3 nM norepinephrine and lower concentrations of epinephrine. It thus appears that growth of C6 cells in serum-containing media desensitizes the beta-adrenergic receptor/cyclic AMP system of these cells. This desensitized state appears to result primarily from the action of catecholamines present in serum. These data indicate that retained catecholamines are one component in serum that can modify expression of beta-adrenergic receptors and hormonal response of cultured glioma cells.     

Structural requirements of the phospholipid substrate for phospholipid N-methylation in rat liver

The role of endogenous phospholipid substrates for phospholipid methylation was investigated in rat liver microsomes. The amount of phosphatidylethanolamine could be drastically reduced by treatment of microsomes with an amino group-blocking compound, methylacetimidate. Simultaneously, the formation of labelled phospholipids from S-adenosyl[Me-3H]methionine decreased, indicating that the amount of endogenous substrate influenced the reaction rate. Phosphatidylmonomethylethanolamine, phosphatidyldimethylethanolamine and phosphatidylmonoethylethanolamine added as dispersions to untreated or treated microsomes stimulated phospholipid methylation, whereas several other phospholipids were inactive. In other experiments the role of phospholipid substrates in intact cells was studied. Cultured rat hepatocytes were enriched in different phospholipids by preincubation with different amino alcohols, and the effects of phospholipid methylation was measured by incubation with [Me-14C]methionine. Phospholipid methylation was significantly stimulated after preincubation with ethanolamine, monomethylethanolamine, monoethylethanolamine and 2-aminobutanol. The results show that both the number and chain length of N-alkyl substituents on phosphatidylethanolamine, as well as other changes in the ethanolamine moiety, will affect the ability of different phospholipids to act as methyl acceptors.     

Nerve Growth Factor Stimulates Phospholipid Methylation in Target Ganglionic Neurons Independently of the Cyclic AMP and Sodium Pump Responses

Suspensions of neurons prepared from embryonic day 12 (E12) chick sympathetic ganglia were incubated with [methyl-3H]methionine in the absence of nerve growth factor (NGF). Presentation of the factor for different periods of time resulted in an approximate three-fold stimulation of radioactivity incorporated into total phospholipid, followed by a rapid decline thereafter. Both the magnitude and the time of the response were dependent on the NGF concentration used. Also examined were possible relationships of phospholipid methylation to two other short-latency responses to NGF, i.e., control of the Na+,K+-pump and elevation of cyclic AMP content. Incubation of E12 sympathetic neurons with known transmethylase inhibitors (shown to be active in the present system) failed to prevent reactivation of the Na+,K+-pump in response to NGF administration. E16 sympathetic neurons and E15 sensory neurons, which do not depend on exogenous NGF for control of their Na+,K+-pump, still show a stimulation of phospholipid methylation when challenged with the factor. Blockage of the pump with ouabain also fails to prevent a methylation response. Thus, the pump and methylation responses to NGF occur independently of each other. Intact E8 chick dorsal root ganglia, but not E12 sympathetic ganglia, display a rapid and transient rise in their cyclic AMP content when presented with NGF. At a concentration of 10 biological units/ml, NGF elicits a peak of phospholipid methylation at 4 min, and a peak of cyclic AMP at 10 min. Methylation inhibitors prevent the methylation response, but not that of cyclic AMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of drugs on lipid methylation,receptor-adenylate cyclase coupling and cyclic AMP secretion in Dictyostelium discoideum

Intercellular communication in Dictyostelium discoideum takes place by means of cyclic AMP-induced cyclic AMP-synthesis and secretion. Since phospholipid methylation has been suggested to play a role in receptor-adenylate cyclase coupling, we examined the effects of transmethylation inhibitors on the production of cyclic AMP during slime mold chemotaxis. Although cycloleucine and l-homocysteic acid were able to inhibit phospholipid methylation and nonpolar lipid methylation up to 40%, these drugs did not cause any decrease of receptor-mediated cAMP-synthesis and secretion. l-Homocysteine thiolactone and dl-homocysteine did not slow down the initial rate of receptor-mediated cAMP-synthesis, but both drugs retarded cAMP-secretion by about 50–75%. Yet, homocysteine thiolactone caused no significant decrease of phospholipid methylation, whereas homocysteine depressed the incorporation of methyl groups into phospholipid up to 80%. Treatment of aggregative cells with 0.1% n-butanol (v/v) resulted in a 3-fold stimulation of phospholipid methylation, whereas nonpolar lipid methylation and cAMP-synthesis were unaffected and cAMP-secretion showed a 2-fold decrease. It is concluded that no direct relationship exists between lipid methylation and receptor-adenylate cyclase coupling or lipid methylation and cAMP-secretion. Homocysteine thiolactone, homocysteine and butanol appear to affect secretion by a mechanism which involves factors other than lipid methylation.     

Reconstitution of beta-adrenergic receptors into phospholipid vesicles: restoration of [125I]iodohydroxybenzylpindolol binding to digitonin-solubilized receptors

beta-adrenergic receptors were solubilized from rat erythrocyte plasma membranes using digitonin. Solubilized receptors were then reconstituted into phospholipid vesicles by the addition of dimyristoylphosphatidylcholine and removal of detergent. Vesicles were separated from residual soluble receptors and detergent by rate-zonal ultracentrifugation. Vesicles were monolamellar, 500-900 A in diameter, and had a lipid content of 6 mumol phospholipid/mg protein. Specific binding of the beta-adrenergic ligand [3H]dihydroalprenolol ([3H]DNA) was 0.9-1.9 pmol/mg protein. Reconstitution of receptors into vesicles restored their ability to bind [125I]iodohydroxybenzylpindolol ([125I]IHYP). This ligand does not bind to detergent-solubilized receptors. [125I]IHYP binding was saturable [Kd = 84 pM] and competed appropriately with (+) and (-) isomers of beta-adrenergic agonists and antagonists. These receptor vesicles therefore appear to be an excellent model system for the study of beta-adrenergic receptor function in a defined lipid milieu.