Effects of chronic exposure to ethanol on the physical and functional properties of the plasma membrane of S49 lymphoma cells

The effects of chronic exposure to ethanol on the physical and functional properties of the plasma membrane were examined with cultured S49 lymphoma cells. The beta-adrenergic receptor-coupled adenylate cyclase system was used as a probe of the functional properties of the plasma membrane. Steady-state fluorescence anisotropy of diphenylhexatriene and the lipid composition of the plasma membrane were used as probes of the physical properties of the membrane. Cells were grown under conditions such that the concentration of ethanol in the growth medium remained stable and oxidation of ethanol to acetaldehyde was not detected. Chronic exposure of S49 cells to 50 mM ethanol or growth of cells at elevated temperature resulted in a decrease in adenylate cyclase activity. There were no changes in the density of receptors or in the affinity of beta-adrenergic receptors for agonists or antagonists following chronic exposure to ethanol. The fluorescence anisotropy of diphenylhexatriene was lower in plasma membranes prepared from cells that had been treated with 50 mM ethanol than in membranes prepared from control cells. However, this change was not associated with changes in the fatty acid composition or the cholesterol to phospholipid ratio of the plasma membrane. There was a small but statistically significant decrease in the amount of phosphatidylserine and an increase in the amount of phosphatidylethanolamine. These changes cannot account for the decrease in anisotropy. In contrast to the effect of ethanol, a decrease in adenylate cyclase activity following growth of S49 cells at 40 degrees C was not associated with a change in anisotropy.     

Desensitization of the beta-adrenergic receptor-coupled adenylate cyclase in cultured mammalian cells. Receptor sequestration versus receptor function

Human A431 and rat glioma C6 cells exposed to isoproterenol underwent a time- and dose-dependent loss of isoproterenol-stimulated adenylate cyclase activity. Desensitization was accompanied by sequestration of beta-adrenergic receptors, which became less accessible to the hydrophilic antagonist 3H-labeled 4-(3-tert-butylamino-2-hydroxypropoxy)benzimidazole-2-one hydrochloride ([3H]CGP-12177) and redistributed from the heavier density plasma membrane fraction to a lighter density membrane fraction. Prior treatment of the cells with concanavalin A or phenylarsine oxide blocked sequestration of the receptors but not desensitization of the agonist-stimulated adenylate cyclase. The membranes from such pretreated cells were exposed to alkali to inactivate adenylate cyclase, and the receptors were transferred to a foreign adenylate cyclase by membrane fusion with polyethylene glycol. beta receptors from desensitized cells exhibited a reduced ability to maximally stimulate the foreign adenylate cyclase, but remained accessible to [3H]CGP-12177 in the fused membranes. When isoproterenol-treated cells were washed free of agonist, there was a time-dependent recovery of agonist responsiveness and [3H]CGP-12177-binding sites. Using the fusion technique, the receptors recovered their functional activity in the resensitized cells. In concanavalin A-treated cells, desensitization and resensitization appeared to occur in the absence of receptor sequestration. Finally, membranes from desensitized cells pretreated with concanavalin A were fused with polyethylene glycol and assayed for agonist-stimulated adenylate cyclase. There was no reversal of the desensitized state. Thus, the primary, essential step in the desensitization process is a reduction in functional activity of the beta-adrenergic receptor. In contrast, sequestration of the receptors is not a prerequisite, but a secondary event during desensitization.     

Association of sequestered beta-adrenergic receptors with the plasma membrane: a novel mechanism for receptor down regulation

Chronic exposure of frog erythrocytes to beta-adrenergic agonists leads to desensitization of the responsiveness of adenylate cyclase to isoproterenol and is accompanied by "down-regulation", a decrease in the number of beta-adrenergic receptors on the cell surface. When frog erythrocyte plasma membranes are prepared by osmotic lysis of cells, the receptors lost from the cell surface during desensitization can be recovered in a "light membrane fraction", obtained by centrifuging the cell cytosol at 158,000 X g for 1 hr. These receptors are sequestered away from the plasma membrane fraction which contains the adenylate cyclase and the guanine nucleotide regulatory protein. If desensitized frog erythrocytes are disrupted by gentler freeze/thaw procedures, however, the sequestered beta-adrenergic receptors can be demonstrated to be physically associated with the plasma membrane. Typically, plasma membranes prepared in this fashion do not demonstrate a significant down regulation despite attenuation of isoproterenol-stimulated adenylate cyclase activity. Under these conditions, beta-adrenergic receptors from control and desensitized preparations co-migrate on sucrose density gradients in exactly the same place as the plasma membrane marker, adenylate cyclase. In contrast, when membranes from osmotically lysed desensitized cells are fractionated on sucrose gradients the down regulated receptors are sequestered in a light membrane fraction which barely enters the gradient and which is physically separated from adenylate cyclase activity. The data are consistent with a novel mechanism of receptor down-regulation which appears to involve the sequestration of the beta-adrenergic receptors away from the cell surface into a membrane compartment which remains physically associated with the plasma membrane.     

Membrane disorder and phospholipid scrambling in electropermeabilized and viable cells

Membrane electropermeabilization relies on the transient permeabilization of the plasma membrane of cells submitted to electric pulses. This method is widely used in cell biology and medicine due to its efficiency to transfer molecules while limiting loss of cell viability. However, very little is known about the consequences of membrane electropermeabilization at the molecular and cellular levels. Progress in the knowledge of the involved mechanisms is a biophysical challenge. As a transient loss of membrane cohesion is associated with membrane permeabilization, our main objective was to detect and visualize at the single-cell level the incidence of phospholipid scrambling and changes in membrane order. We performed studies using fluorescence microscopy with C6-NBD-PC and FM1-43 to monitor phospholipid scrambling and membrane order of mammalian cells. Millisecond permeabilizing pulses induced membrane disorganization by increasing the translocation of phosphatidylcholines according to an ATP-independent process. The pulses induced the formation of long-lived permeant structures that were present during membrane resealing, but were not associated with phosphatidylcholine internalization. These pulses resulted in a rapid phospholipid flip/flop within less than 1 s and were exclusively restricted to the regions of the permeabilized membrane. Under such electrical conditions, phosphatidylserine externalization was not detected. Moreover, this electrically-mediated membrane disorganization was not correlated with loss of cell viability. Our results could support the existence of direct interactions between the movement of membrane zwitterionic phospholipids and the electric field.     

Specific electropermeabilization of leucocytes in a blood sample and application to large volumes of cells

Electropermeabilization is obtained when the membrane potential difference reaches a critical threshold. This is performed by submitting cells to an external electric field pulse. The field modulates the endogenous potential difference in a cell-size-dependent way. Computer simulations predict that large cells would be specifically permeabilized in a mixture with smaller cells. This was examined on a mixture of Chinese hamster ovary (CHO) cells and erythrocytes. CHO cells were permeabilized to Trypan blue without any occurrence of haemolysis. A similar 'size' specificity was observed on blood samples. This agreement between prediction and experimental observation indicates that induction of electropermeabilization is mainly under the control of the size of the target cell. Its physiology plays only a minor role, if any. Treating blood with 10 square wave pulses lasting 100 microseconds of an intensity of 1.6 kV/cm induced the permeabilization of 70% of the leucocytes (polymorphs and monocytes) but did not affect erythrocytes. No washing of the sample was needed in a procedure in which cells were pulsed in the plasma. A flow electropulsing process allows the treatment of large blood volumes under conditions where cells are kept viable. These results show that electropermeabilization could be used as an effective way to obtain immunocompatible drug vehicles.     

Isolation of adenylate cyclase-enriched membranes from mammalian cells using concanavalin A.

Plasma membrane vesicles containing adenylate cyclase and beta-adrenergic receptors were prepared from 1321N1 human astrocytoma cells by a procedure involving the use of concanavalin A to stabilize the plasma membrane to fragmentation and vesiculation upon cell lysis. Treatment of cells with concanavalin A causes these plasma membrane markers to sediment to a higher density of sucrose and in a narrower band than observed with untreated cells. Upon treatment of the heavy membrane fragments with alpha-methylmannoside to remove bound concanavalin A, the enzyme markers again sediment a lower densities of sucrose. This reversible change in sedimentation behavior has been used to obtain preparations of plasma membranes enriched 14- to 21-fold (recovery 25%) in adenylate cyclase activity and about 12-fold (recovery 16%) in beta-adrenergic receptor density, as compared to lysates. The adenylate cyclase of purified membranes responded normally to isoproterenol and prostaglandin E1. Experiments with S49 and YAC mouse lymphoma cells and human skin fibroblasts indicate that this procedure may be adaptable to the isolation of plasma membranes from a variety of cultured cell lines.     

Characterization of an altered membrane form of the beta-adrenergic receptor produced during agonist-induced desensitization

Incubation of 1321N1 human astrocytoma cells with 1 microM isoproterenol rapidly results in the conversion of a portion of the beta-adrenergic receptors to a membrane form that can be separated from markers for the plasma membrane by sucrose density gradient or differential centrifugation. This "light peak" form of the receptor reaches a maximal level within 10 min of incubation of cells with catecholamine. Two types of experiments suggest that the early phase of catecholamine-induced desensitization of the beta-adrenergic receptor-linked adenylate cyclase can be separated into at least two reactions. First, the agonist-induced loss of catecholamine-stimulated adenylate cyclase activity precedes the appearance of beta-adrenergic receptors in the light peak fraction by 1-2 min. Second, pretreatment of cells with concanavalin A prior to induction of desensitization blocks the formation of the light peak form of beta-adrenergic receptors without blocking the "uncoupling" reaction as measured by catecholamine-stimulated adenylate cyclase activity. Specificity for the reaction that converts beta-adrenergic receptors to the light peak form is indicated by the lack of a catecholamine-induced alteration in the sucrose density gradient distribution of muscarinic cholinergic receptors, adenylate cyclase or the guanine nucleotide-binding proteins, Ns and Ni. The light peak of beta-adrenergic receptors migrates at a density similar to that of at least a portion of the activity of galactosyltransferase, a marker for Golgi. Enzyme marker activities for lysosomes and endoplasmic reticulum are not associated with this population of beta-adrenergic receptors. Taken together, these and other data suggest that incubation of 1321N1 cells with isoproterenol results in a rapid uncoupling of beta-adrenergic receptors from adenylate cyclase which is followed by a change in the membrane form of the receptor. This latter step most likely represents internalization of receptors into a vesicular form which may then serve as the precursor state from which receptors are eventually lost from the cell.     

Exfoliation of the beta-adrenergic receptor and the regulatory components of adenylate cyclase by cultured rat glioma C6 cells

Cultured rat glioma C6 cells exfoliate membrane vesicles which have been termed 'exosomes' into the culture medium. The exosomes contained both stimulatory and inhibitory GTP-binding components of adenylate cyclase (the stimulatory, Gs, and the inhibitory, Gi, regulatory components) and beta-adrenergic receptors but were devoid of adenylate cyclase activity. It was therefore apparent that the catalytic component of adenylate cyclase was either not exfoliated or was inactivated during the exfoliation process. The presence of Gs or Gi in the exosomes was detected by ADP ribosylation using [alpha-32P]NAD in the presence of cholera or pertussis toxins, respectively. The exosomal concentration of each of the two components was estimated to be about one fifth of that of the cell membrane when expressed on a per mg protein basis. Exosomal Gs was almost as active as the membrane-derived Gs in its ability to reconstitute NaF- and guanine nucleotide-stimulated adenylate cyclase activity in membranes of S49 cyc- cells, which lack a functional Gs. The ability of exosomal Gs to reconstitute isoproterenol-stimulated activity, however, was much lower than that of membrane Gs. The density of beta-adrenergic receptors in the exosomes was much less than that found in the membranes. Although the exosomal receptors bound the antagonist iodocyanopindolol with the same affinity as receptors from the cell membrane, the affinity for the agonist isoproterenol was 13- to 18-fold lower in the exosomes. In addition, this affinity was not modulated by GTP in the exosomes. Thus, exfoliated beta-adrenergic receptors seem to be impaired in their ability to couple to and activate Gs. This was directly tested by coupling the receptors to a foreign adenylate cyclase using membrane fusion. The fusates were then assayed for agonist-stimulated activity. While significant stimulation of the acceptor adenylate cyclase was obtained using C6 membrane receptors, the exosomal receptors were completely inactive. Thus during exfoliation, there appear to be changes in the components of the beta-adrenergic-sensitive adenylate cyclase that results in a nonfunctional system in the exosomes.     

Fluorescent localization of the beta-adrenergic receptor on DDT-1 cells. Down-regulation by adrenergic agonists.

Continuous incubation of cultured cells with beta-adrenergic agonists results in the desensitization of adrenergic responsiveness accompanied by the down-regulation of cell surface beta-adrenergic receptors (beta AR). Previous studies have relied on measurements of ligand binding activity for the detection of the beta AR in the cell. In the present study, we have raised a monoclonal antibody to a synthetic peptide corresponding to amino acid numbers 226-239 of the hamster beta 2AR. This antibody was used to localize the beta AR in hamster smooth-muscle DDT-1 cells by immunofluorescence, without regard for the ability of the receptor to bind ligands. The beta AR was found to be localized primarily at the plasma membrane of these cells, with a nonhomogeneous pattern of distribution. A rapid loss of beta AR-specific immunofluorescence, which paralleled receptor down-regulation as measured by ligand-binding activity, was seen with beta-adrenergic agonists, but not with antagonists. In addition, a transient increase in fluorescence was observed after short times of exposure of the cells to agonists. This fluorescence increase may reflect a ligand-induced conformational change in the receptor.     

Determination of the desensitization of beta-adrenergic receptors by [3H]CGP-12177.

Isoprenaline treatment of C6-glioma cells induced a fast decrease in the number of beta-adrenergic receptors as determined by binding of [3H]CGP-12177, which paralleled the decrease in the hormonally stimulated adenylate cyclase activity. The total number of receptors, as determined by binding of (-)-[3H]dihydroalprenolol, did not decrease. Separation of the beta-adrenergic receptors on a sucrose density gradient showed that the decrease in the number of receptors detectable with CGP-12177 was due to a movement of the receptors from the plasma membrane to a vesicular cell compartment. By using both (-)-[3H]dihydroalprenolol and [3H]CGP-12177 it is thus possible to differentiate between the total number of receptors and those present at the plasma membrane in an unfractionated cell lysate.     

Experimental conditions influence [3H]-dihydroalprenolol binding characteristics to living HeLa cells due to morphological changes: a warning

Harvesting of plated growing HeLa cells, followed by incubation of these cells without any addition at 37 degrees C was found to cause changes in the cell shape. This phenomenon is accompanied by a diminished binding of the beta-adrenergic antagonist [3H]-dihydroalprenolol and the alpha-adrenergic antagonist phentolamine to a binding compartment not representing beta-adrenergic receptors. These binding sites have a high affinity for hydrophobic agents and most probably represent lipophilic structures in the cellular membrane. Changes in the cell shape obviously cause alterations in the physical properties of the plasma membrane. This might lead to misinterpretations of the results from experiments in which the redistribution of beta-adrenergic receptors is followed during incubation with agonists, as receptor occupation with subsequent receptor redistribution is possibly accompanied by effects on the membrane microviscosity. It is concluded that investigations performed in order to follow physiological events like receptor redistribution and desensitization processes, may be obfuscated by changes in the normal physical state of the living cells.     

Uptake of L-[propyl-2,3-3H]dihydroalprenolol by intact HeLa cells

This report describes the uptake of L-[propyl-2,3-3H]dihydroalprenolol, a beta-adrenergic antagonist, by HeLa (human adenocarcinoma) cells. [3H]Dihydroalprenolol binds to sites of high capacity and low affinity in intact HeLa cells. The binding achieves equilibrium rapidly and is rapidly reversible. Bound [3H]dihydroalprenolol is displaceable by beta-adrenergic antagonists in a nonstereoselective fashion, but is not displaceable by isoproterenol, an adrenergic agonist. Phentolamine, an alpha-adrenergic antagonist, and chloroquine, a lysosomotropic amine, also compete for [3H]dihydroalprenolol binding sites. [3H]Dihydroalprenolol binding is inhibited by metabolic inhibitors, but not by cytoskeletal blocking agents. The binding is sensitive to extracellular pH (less binding at lower pH) and is temperature-sensitive (less binding at lower temperatures). The bound radioligand is rapidly reversed following hypotonic lysis of the cells. These [3H]dihydroalprenolol binding sites in intact HeLa cells therefore do not have the characteristics expected for beta-adrenergic receptors. Further studies showed that beta-adrenergic receptors could be detected in a HeLa membrane preparation using [125I]iodohydroxybenzylpindolol, and that chloroquine had very low affinity for these receptors. We conclude that [3H]dihydroalprenolol diffuses across the plasma membrane of intact HeLa cells and accumulates in acidic intracellular compartments.     

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.     

Characteristics of the beta-adrenergic adenylate cyclase system of developing rabbit bone-marrow erythroblasts.

After fractionation of rabbit bone marrow into dividing (early) and non-dividing (late) erythroid cells, the adenylate cyclase activity of membrane ghosts was assayed in the presence of guanine nucleotides ((GTP and its analogue p[NH]ppG (guanosine 5'-[beta, gamma-imido]triphosphate))), the beta-adrenergic agonist L-isoprenaline (L-isoproterenol) and the antagonist L-propranolol. Both GTP and p[NH]ppG increased the adenylate cyclase activity of early and late erythroblasts, whereas the stimulating effect of the beta-adrenergic drug L-isoprenaline was limited to the immature dividing bone-marrow cells. The effect of L-isoprenaline was completely inhibited by the antagonist L-propranolol, confirming that the response was due to stimulation of beta-adrenergic receptors on the plasma membrane. The lack of response of non-dividing erythroblasts to beta-adrenergic stimuli is not due to loss of beta-receptors, since both dividing and non-dividing cells bind the selective ligand [125I]iodohydroxybenzylpindolol with almost equal affinities, the apparent dissociation constants, Kd, being 0.91 X 10(-8)M and 1.0 X 10(-8) M respectively. The number of beta-adrenergic receptors per cell was 2-fold higher in the dividing cells. No significant change in binding affinity for GTP and p[NH]ppG during erythroblast development was observed: the dissociation constants of both guanine nucleotides were almost identical with early and late erythroblast membrane preparations [2-3 (X 10(-7) M]. With dividing cells, however, in the presence of L-isoprenaline the dissociation constants of GTP and p[NH]ppG were lower (6 X 10(-8) M). The dose-response curves for isoprenaline competition in binding of [125I]iodohydroxybenzylpindolol by dividing cells showed that the EC50 (effective concentration for half maximum activity) value for isoprenaline was higher in the presence of p[NH]ppG. With non-dividing cells the EC50 value for isoprenaline was equal in the presence and in the absence of p[NH]ppG and similar to that observed with dividing-cell membranes in the presence of the nucleotide. Thus differentiation of rabbit bone-marrow erythroid cells seems to be accompanied by uncoupling of the beta-adrenergic receptors from the adenylate cyclase catalytic protein as well as by a decrease in the number of receptors per cell, but not by changes in the catecholamine and guanine-nucleotide-binding affinities.     

The hepatic adrenergic receptors

Summary The presence of both alpha- and beta-adrenergic receptors in liver designated the hepatic plasma membrane as a useful tool for the elucidation of the mechanisms by which the hormonal signal is transfered through the membrane via a coupling system to an amplifying entity. This process is well documented for the beta-adrenergic receptor which is linked to adenylate cyclase, whereby it modulates the cyclic AMP level. Much less is known about the alpha-adrenergic receptor.Recently, two factors have contributed to a renewed interest in alpha- and beta-adrenergic receptors in liver: i) The fact that activation of glycogenolysis in isolated liver parenchymall cells by epinephrine may be mediated by either alpha- or beta-adrenergic receptors, depending on the species or on the state of nutrition, and not only by beta-adrenergic receptors as previously thought. ii) The existence of specific adrenergic agonists and antagonists radiolabeled to a high specific activity which has permitted the characterization of adrenergic receptors in terms of nature, number, affinity and regulation.The present review will be devoted to the recent progress made in the physiological, pharmacological and biochemical characterization of alpha- and beta-adrenergic receptors in the liver.     

Regulation of beta-adrenergic receptors and calcium channel agonist binding sites in cultured human embryonal smooth muscle cells

Recent electrophysiological studies with cell membrane patches of cardiac myocytes and an electrically excitable cell line derived from rat pituitary tumor suggested that voltage activated calcium channels must be phosphorylated to respond to membrane depolarization (Armstrong and Eckert 1986; Trautwein and Kameyama 1986). In view of the "phosphorylation hypothesis" we investigated the adenylate-cyclase activity, the characteristics of beta-adrenergic and calcium channel agonist binding sites in control and desensitized (exposure to isoproterenol) human embryonal cells (HEC), and in fragmented membrane preparations of canine coronary smooth muscle. Our results suggest that down-regulation of the membrane-bound beta-adrenergic receptors, induced by isoproterenol in human embryonal cells and also in adult canine vascular tissue, results in physical translocation of beta-adrenergic binding sites into the light membrane fraction. This phenomenon is accompanied with an increased intracellular concentration of cAMP in and an increased binding of the calcium channel agonist (3H) BAYK 8644 to both HEC and canine smooth muscle membrane preparations. It could be concluded that phosphorylation of beta-adrenergic receptors regulates not only the beta subcellular distribution of the beta receptors but also the availability of calcium channel agonist binding sites in the cellular membrane.     

Cholesterol modulation of membrane fluidity and VIP receptor/effector system in rat prostatic epithelial cells.

Treatment of rat prostatic epithelial cells with cholesteryl hemisuccinate (ChH) resulted in a time- and dose-dependent inhibition of the stimulatory effect of the neuropeptide vasoactive intestinal peptide (VIP) on cyclic AMP accumulation, with a 40% decrease in the response to a maximally effective VIP concentration. Cell treatment with ChH led also to a similar blocking of isoproterenol (a beta-adrenergic agonist) action but did not modify forskolin (which is assumed to act directly on the catalytic unit of adenylate cyclase) activity upon cyclic AMP levels. The levels of the transduction protein Gs were similar in membranes from both control and ChH-treated cells as suggested by experiments on cholera toxin-catalyzed ADP-ribosylation. The inhibitory effect of ChH was accompanied by an increase of membrane microviscosity as estimated by measurements of fluorescence polarization. Experiments on VIP binding indicated that increasing cholesterol concentration in the plasma membrane led to a higher VIP binding capacity without changes in the affinity of VIP receptors. These data suggest that membrane cholesterol incorporation diminishes the coupling efficiency between adenylate cyclase and the VIP-receptor complex or other receptor systems (i.e., desensitization) due to an increase of plasma membrane rigidity.     

Inverse regulation of calcium channels and beta-adrenergic receptors in virus-transformed human embryonal cells.

Monolayer cultures of human embryonal smooth muscle cells (HEC) were used to study the heterologous regulation of membrane beta-adrenergic receptors and Ca2+ channels. The relationships between the activation of membrane bound alpha-1 and beta-adrenergic receptors, the cyclic nucleotide response and Ca2+ channel binding were studied in a cellular model of latent virus infection (Herpes simplex, Type-2) in a human embryonal cell line. In the early stage of infection (72 h), there was a significant increase in the cell cAMP content, followed by a decrease in the binding capacity of the beta-adrenergic ligand with an increased total number of the 1,4-dihydropyridine Ca2+ channel agonist (-)-S-(3H)BAYK 8644 binding sites on the cell membrane of infected cells. The increased numbers of Ca2+ agonist binding sites were accompanied by an increased cAMP content in the cells and an increased membrane ATP-ase activity. Down-regulation of (3H)DHA binding, and an increased capacity of Ca2+ agonist binding were found after prolonged exposure of HEC to isoprenaline (10(-5) mol.l-1). Stimulation of alpha-1 adrenergic receptors with phenylephrine (10(-6) mol.l-1) was accompanied with only slight but significant increase in (3H)DHA binding and with a significant reduction in the total number of Ca2+ channel agonist binding sites.     

Somatostatin induces translocation of the beta-adrenergic receptor kinase and desensitizes somatostatin receptors in S49 lymphoma cells

The beta-adrenergic receptor kinase is a cytosolic enzyme that specifically phosphorylates the agonist-occupied form of the beta-adrenergic receptor (beta AR). Beta AR kinase appears to be translocated from the cytosol to the plasma membrane when kin- S49 lymphoma cells are incubated with either beta-adrenergic agonists or prostaglandin E1, both of which act through receptors which stimulate adenylate cyclase. We report here that brief (approximately 20 min) exposure of wild type S49 lymphoma cells to somatostatin (which inhibits adenylate cyclase) promotes the translocation of beta AR kinase to an extent comparable to that observed in the presence of the beta agonist isoproterenol or prostaglandin E1. Beta AR kinase activity can be measured using either beta AR or rhodopsin, the retinal receptor for light, as a substrate. The translocation process triggered by somatostatin is rapid, reversible, and is associated with somatostatin receptor desensitization. The latter is apparent as an attenuation of the inhibition by somatostatin of forskolin-stimulated adenylate cyclase activity in membranes of S49 cells preincubated in the presence of the peptide. These results strongly suggest that beta AR kinase is able to phosphorylate and desensitize both stimulatory and inhibitory adenylate cyclase-coupled receptors, thus emerging as a general kinase that regulates the function of different receptors in an agonist-specific fashion.     

Colocalization of beta-adrenergic receptors and caveolin within the plasma membrane.

The rapid amplification of beta-adrenergic receptor signaling involves the sequential activation of multiple signaling molecules ranging from the receptor to adenylyl cyclase. The prevailing view of the agonist-induced interaction between signaling molecules is based on random collisions between proteins that diffuse freely in the plasma membrane. The recent identification of G protein alpha- and betagamma-subunits in caveolae and their functional interaction with caveolin suggests that caveolae may participate in G protein-coupled signaling. We have investigated the potential interaction of beta-adrenergic receptors with caveolin under resting conditions. beta1- and beta2-adrenergic receptors were recombinantly overexpressed in COS-7 cells. Caveolae were isolated using the detergent-free sucrose gradient centrifugation method. beta1- and beta2-adrenergic receptors were localized in the same gradient fractions as caveolin, where Gsalpha- and betagamma-subunits were detected as well. Immunofluorescence microscopy demonstrated the colocalization of beta-adrenergic receptors with caveolin, indicating a nonrandom distribution of beta-adrenergic receptors in the plasma membrane. Using polyhistidine-tagged recombinant proteins, beta-adrenergic receptors were copurified with caveolin, suggesting that they were physically bound. Our results suggest that, in addition to clathrin-coated pits, caveolae may act as another plasma membrane microdomain to compartmentalize beta-adrenergic receptors.