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The Cascadia subduction zone (CSZ) is an exceptional geologic environment for recording evidence of land-level changes, tsunamis, and ground motion that reveals at least 19 great megathrust earthquakes over the past 10 kyr. Such earthquakes are among the most impactful natural hazards on Earth, transcend national boundaries, and can have global impact.Reducing the societal impacts of future events in the US Pacific Northwest and coastal British Columbia, Canada, requires improved scientific understanding of megathrust earthquake rupture, recurrence, and corresponding hazards. Despite substantial knowledge gained from decades of research, large uncertainties remain about the characteristics and frequencies of past CSZ earthquakes. In this review, we summarize geological, geophysical, and instrumental evidence relevant to understanding megathrust earthquakes along the CSZ and associated uncertainties. We discuss how the evidence constrains various models of great megathrust earthquake recurrence in Cascadia and identify potential paths forward for the earthquake science community. ▪ Despite outstanding geologic records of past megathrust events, large uncertainty of the magnitude and frequency of CSZ earthquakes remains. ▪ This review outlines current knowledge and promising future directions to address outstanding questions on CSZ rupture characteristics and recurrence. ▪ Integration of diverse data sets with attention to the geologic processes that create different records has potential to lead to major progress.

Bedrock weathering and soil production set the rate that hillslope colluvium is produced, while material properties determine whether sediment can fail under a given set of conditions. Together the...

The Altiplano-Puna Magma Body (APMB) in the Central Andes is the largest imaged magma reservoir on Earth, and is located within the second highest orogenic plateau on Earth, the Altiplano-Puna. Although the APMB is a first-order geologic feature similar to the Sierra Nevada batholith, its role in the surface uplift history of the Central Andes remains uncertain. Here we show that a long-wavelength topographic dome overlies the seismically measured extent of the APMB, and gravity data suggest that the uplift is isostatically compensated. Isostatic modelling of the magmatic contribution to dome growth yields melt volumes comparable to those estimated from tomography, and suggests that the APMB growth rate exceeds the peak Cretaceous magmatic flare-up in the Sierran batholith. Our analysis reveals that magmatic addition may provide a contribution to surface uplift on par with lithospheric removal, and illustrates that surface topography may help constrain the magnitude of pluton-scale melt production., The Altiplano-Puna magma body is located in the world's second highest plateau, the Altiplano-Puna, but the influence of melt production in the surface uplift of the Central Andes is unclear. Perkins et al. link surface topography and isotactic modelling to constrain the melt production in the magma body.

Geodetic surveys of Volcan Uturuncu and the Lazufre volcanic complex in the Central Andes of South America reveal sustained surface uplift from magmatic intrusion at depth. However, the decadal timescales of geodetic surveys are short relative to the timescales of magma chamber growth. Thus, from ­geodesy alone, it is difficult to infer the deformation and hence magma accumulation history of these volcanoes. Here we combine data from InSAR, long-wavelength topography, GPS and high-resolution topographic surveys of lake shorelines and rivers, and lava flow morphology to constrain the spatial and temporal evolution of magmatism at Uturuncu and Lazufre. Near Uturuncu, dated lake shorelines show no evidence of tilting since ca. 16 ka, and we find no evidence of deformation in the long-wavelength topography. A lack of net surface displacement suggests that uplift related to a rising diapir must be less than a century old, or, more likely, magmatic inflation at Uturuncu is transient over millennial timescales and is therefore not recorded in the topog­raphy. At Lazufre, we also find no evidence for sustained uplift recorded in Late Pleistocene lake shorelines. However, the orientations of multiple dated lava flows suggest that the long-wavelength dome at the center of Lazufre’s uplift has persisted since at least 400 ka. Additionally, we find that the radial distribution of volcanic vents at Lazufre, coupled with the presence of an apical graben, is consistent with experimental and theoretical predictions of magmatic doming. The dome’s longevity indicates significant magma storage at depth, and therefore Lazufre is likely a highly evolved pre-caldera magmatic system. These two case studies demonstrate that combining geomorphic and geophysical data sets to extend the geodetic record back in time can help determine the style and magnitude of magma transport in volcanic systems.

Researchers examine the rings of drowned trees in landslide-dammed lakes for clues to today’s earthquake hazards in the Pacific Northwest.

The Central Andes is a key global location to study the enigmatic relation between volcanism and plutonism because it has been the site of large ignimbrite- forming eruptions during the past several million years and currently hosts the world's largest zone of silicic partial melt in the form of the Altiplano- Puna Magma (or Mush) Body (APMB) and the Southern Puna Magma Body (SPMB). In this themed issue, results from the recently completed PLUTONS project are synthesized. This project focused an interdisciplinary study on two regions of large-scale surface uplift that have been found to represent ongoing movement of magmatic fluids in the middle to upper crust. The locations are Uturuncu in Bolivia near the center of the APMB and Lazufre on the Chile-Argentina border, on the edge of the SPMB. These studies use a suite of geological, geochemical, geophysical (seismology, gravity, surface deformation, and electromagnetic methods), petrological, and geomorphological techniques with numerical modeling to infer the subsurface distribution, quantity, and movements of magmatic fluids, as well as the past history of eruptions. Both Uturuncu and Lazufre show separate geophysical anomalies in the upper, middle, and lower crust (e.g., low seismic velocity, low resistivity, etc.) indicating multiple distinct reservoirs of magma and/or hydrothermal fluids with different physical properties. The characteristics of the geophysical anomalies differ somewhat depending on the technique used-reflecting the different sensitivity of each method to subsurface melt (or fluid) of different compositions, connectivity, and volatile content and highlight the need for integrated, multidisciplinary studies. While the PLUTONS project has led to significant progress, many unresolved issues remain and new questions have been raised.

Water is considered the primary agent that erodes and shapes bedrock canyons. Analyses of canyon morphology in the central Andes suggest that abrasion by wind can amplify canyon incision and reshape canyons on Earth—and possibly on Mars.

In many cells catecholamines induce a translocation of beta-adrenergic receptors from the cell surface to intracellular vesicular sites. We have postulated that the translocation event is the result of ligand-induced endocytosis of the receptor, probably via clathrin-coated pits. Previously, we demonstrated that reduction of cellular ATP content with antimycin A completely blocked endocytosis of epidermal growth factor and translocation of beta-adrenergic receptors in 1321N1 astrocytoma cells. However, the effect of reduction in ATP content on endocytosis remains controversial. In the present report, we demonstrate that reduction of ATP content to a level5% of that in control cells is sufficient to prevent endocytosis of [125I]iodotransferrin and translocation of beta-adrenergic receptors. The further demonstration that reactions leading to the return of internalized transferrin or beta-adrenergic receptors to the cell surface are blocked after relatively modest reductions in ATP content provides further evidence of the similarity in the processes subserving diacytosis of beta-adrenergic receptors and transferrin. The differential requirement for ATP of the two arms of diacytosis provides the basis for an explanation of the controversy regarding a requirement for ATP in endocytosis via clathrin-coated pits.

The kinetics of the return of internalized beta-adrenergic receptors to the plasma membrane were measured in human astrocytoma cells. The movement of [125I]iodopindolol-labeled receptors back to the plasma membrane was measured directly and was shown to occur with a t1+2 of 3-4 min. Unlabeled receptors appeared to exhibit the same kinetics of externalization. The process was not inhibited by low concentrations (1-10 microM) of propranolol or even high concentrations of isoproterenol (0.1-1.0 mM). Higher concentrations of propranolol (0.1-1.0 mM) and other lipophilic amines inhibited externalization. The results are consistent with the proposal that catecholamine-induced beta-adrenergic receptor internalization and externalization (diacytosis) occur via the clathrin-coated pit/endosome pathway.

Recombinant turkey erythrocyte beta-adrenergic receptors expressed in murine L cells exhibited characteristic avian subtype selectivity for agonists and antagonists. In 10 of the 11 clones studied, no agonist-induced internalization of receptor was observed, although agonist-induced uncoupling of receptor and adenylyl cyclase occurred rapidly. GTP caused little or no decrease in affinity for beta-adrenergic agonists. Such behavior is commonly observed in avian erythrocytes. In contrast, one clone was susceptible to agonist-induced receptor internalization and down-regulation even though it exhibited characteristic avian beta-adrenergic ligand-binding properties. The affinity of this variant receptor for agonists was also notably reduced by GTP. Electrophoresis of affinity-labeled receptor from this clone indicated an apparent size of about 33 kDa, about 12 kDa less than that of the native or recombinant turkey beta-adrenergic receptor. Genomic DNA from this cell line that encodes the receptor was cloned and partially sequenced. The coding region of the original receptor cDNA was interrupted after codon 412 (out of 483) and was followed by 36 base pairs of novel sequence prior to the first in-frame stop codon. These results suggest that the lack of both hormone-induced internalization and GTP-sensitive, high affinity binding of agonists that is characteristic of the beta-adrenergic receptor in avian erythrocytes is due to intrinsic properties of the receptor. The restoration of these phenomena in a C-terminally truncated mutant receptor suggests the importance of the C-terminal domain in determining these processes.

Competition by beta-adrenergic agonists and antagonists for 125I-pindolol binding sites on intact cells (1321N1 human astrocytoma and C62B rat glioma) was measured using short time binding assays as previously described (Toews, M. L., Harden, T. K., and Perkins, J. P. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3553-3557). Preincubation of cells with agonists converted about half of the cellular beta-adrenergic receptors from a form exhibiting high affinity for the agonists isoproterenol and epinephrine and the antagonist sotalol to a form exhibiting much lower apparent affinity for these ligands in short time assays. Exposure to agonists did not alter the affinity of receptors for the antagonist metoprolol. This change in the ligand binding properties of the receptor was rapid (t1/2 = 1-2 min following a lag of about 0.5 min), reversible (t1/2 = 6-8 min), and dependent on the agonist concentration present during the preincubation (K0.5 = 15 nM for isoproterenol). Both isoproterenol and sotalol attained equilibrium with the high affinity receptors very rapidly but equilibrated only slowly with those receptors exhibiting low apparent affinity in short time assays. These results are interpreted in terms of a model which postulates that both the low apparent affinity in short time assays and the subsequent slow equilibration of hydrophilic ligands with these receptors result from agonist-induced internalization of a fraction of cell surface beta-adrenergic receptors. The relationship of this change in receptor binding properties to other aspects of agonist-induced desensitization of the beta-adrenergic receptor-coupled adenylate cyclase system is discussed.

Beta-Adrenergic receptors and epidermal growth factor receptors are both expressed on the cell surface of human astrocytoma cells. Incubation with a catecholamine or epidermal growth factor results in rapid internalization of the respective receptor. The internalized receptors co-migrate in light fractions on sucrose gradients. Astrocytoma cells maintain a constant ATP concentration by either glycolytic or mitochondrial ATP production. When cells are incubated in a medium depleted of substrates for glycolysis and gluconeogenesis, addition of inhibitors of mitochondrial ATP synthesis causes a rapid reduction in cellular ATP content. An immediate return to control ATP levels occurs upon addition of an appropriate nutrient, such as glucose. Decreasing the cellular ATP content to less than 10% of control markedly inhibits internalization of beta-adrenergic receptors and epidermal growth factor. The inhibition of endocytosis is reversed as soon as the intracellular ATP content is restored. Previous work by others (Clarke, B.L., and Weigel, P.H. (1985) J. Biol. Chem. 260, 128-133) suggested that ATP is not required for internalization (per se) of asialoglycoprotein in hepatocytes but was required for recycling of the asialoglycoprotein receptor. In contrast, our results indicate that in astrocytoma cells the process of internalization of epidermal growth factor and beta-adrenergic receptors, per se, is highly ATP dependent.

Two-dimensional polyacrylamide gel electrophoresis and the radioligand (-)-[125I]iodopindolol (125I-Pin) have been used to study isoproterenol-dependent protein phosphorylation and beta-adrenergic receptor availability, respectively, in cultured Sertoli cells and freshly isolated seminiferous tubular segments of sexually immature and mature rats. Sertoli cells prepared from sexually immature rats show progressive 125I-Pin binding in primary cultures that correlates with isoproterenol-induced cell shape changes, redistribution of immunoreactive vimentin, and phosphorylation of this intermediate filament protein. The development of 125I-Pin binding to Sertoli cell lysates is blocked by cycloheximide. Seminiferous tubules do not show significant isoproterenol-dependent vimentin phosphorylation nor 125I-Pin binding. However, vimentin phosphorylation can be induced by follicle-stimulating hormone or a cyclic nucleotide analog. This study stresses the need for correlating pharmacological-induced responses observed in Sertoli cell primary cultures with those in the intact seminiferous tubule.

The redistribution of beta-adrenergic receptors (beta-AR) during agonist-induced desensitization has been compared to the process of receptor-mediated endocytosis of epidermal growth factor (EGF) in human astrocytoma cells (1321N1). [125I]EGF exhibited saturable binding to high affinity (KD = 1-2 nM) receptor sites on intact 1321N1 cells. [125I]EGF was found to internalize rapidly using an acid wash technique to remove surface bound hormone. Sucrose density gradient fractionation following exposure to EGF revealed a redistribution of EGF binding sites from high density (heavy peak) to low density (light peak) regions of the gradient. The light peak binding probably represents EGF in internalized vesicles formed during endocytosis. Low temperature (4 degrees C) or the presence of the lectin concanavalin A (con A) inhibited this ligand-induced movement of EGF receptors. When cells were incubated simultaneously with EGF and the beta-AR agonist isoproterenol, both receptors were found to co-migrate in the low density regions of sucrose gradients. No evidence of heterologous ligand-induced receptor endocytosis was found. These results suggest that the EGF receptors and beta-AR are processed in parallel by 1321N1 cells.

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.

Incubation of human astrocytoma cells (1321N1) with low concentrations of isoproterenol results in a specific loss of responsiveness to catecholamines as evidenced by a decreased accumulation of cAMP in intact cells, a reduction in isoproterenol-stimulated adenylate cyclase activity, and a decrease in beta-adrenergic receptor density, as measured by the specific binding of 125I-hydroxybenzylpindolol. The kinetics of desensitization suggest the involvement of two different reactions. The initial reaction involves a rapid loss of adenylate cyclase activity with little loss of beta-adrenergic receptors. Subsequently, a slower reaction results in the loss of measurable beta-adrenergic receptors. The degree of loss of both parameters was similar after 24 h of desensitization. It is concluded that the loss of beta-adrenergic receptors is an event that occurs as a result of the initial uncoupling of the beta-receptor-linked adenylate cyclase.

The ligand-induced internalization of beta-adrenergic receptors and the receptor-mediated internalization of epidermal growth factor were blocked, under similar conditions, by phenylarsine oxide (PAO) in human astrocytoma cells (1321N1). The inhibition was not prevented or reversed by monofunctional sulfhydryl agents such as 2-mercaptoethanol or glutathione; however, the inhibitory action of PAO was blocked and reversed by bifunctional thiols such as 2,3-dimercaptoethanol or dithiothreitol. The results are consistent with the interaction of PAO with vicinal sulfhydryl groups to form a stabile ring structure. PAO did not prevent isoproterenol-induced uncoupling (desensitization) of beta-adrenergic receptors even though receptor internalization was completely blocked. The effects of PAO on receptor internalization could not be explained by any action of the trivalent arsenical to lower ATP levels. Ligand binding to both receptors was not detectably altered by PAO under conditions selective for inhibition for endocytosis. The results suggest a common mechanism for internalization of beta-adrenergic receptors and epidermal growth factor by a process that involves vicinal sulfhydryl groups.

Prostaglandins (PG) of the E series and catecholamines stimulate adenosine 3':5'-monophosphate (cAMP) formation in human astrocytoma cells (1321N1). These two classes of effectors activated adenylate cyclase upon interaction with different receptor systems. No evidence for a mediatory role for PG in the action of catecholamines was found. PG interacted with 1321N1 cells with an order of potency of PGE1 = PGE2 greater than PGA1 greater than PGF2 alpha. The effect of combinations of the various PG indicated that all efficacious PG interacted with a common receptor. 7-Oxa-13-prostynoic acid and indomethacin were shown to be competitive inhibitors of the effect of PGE1 with Ki values of 4 and 150 micron, respectively. These two compounds did not inhibit the effect of isoproterenol. Polyphloretin phosphate caused a complex pattern of inhibition of the effects of PGE1 and at higher concentrations also inhibited the effects of isoproterenol. The mefenamate class of nonsteroidal anti-inflammatory agents was found to inhibit the effects of PGE1 with a potency order of meclofenamic acid greater than flufenamic acid = mefenamic acid. The inhibitory action of meclofenamic acid was complex involving specific, but partial, insurmountable antagonism of PGE1 as well as competitive inhibition of PGE1 effects. At higher concentrations of meclofenamic acid a nonspecific inhibition of the effects of both PGE1 and isoproterenol was observed. These studies suggest that the inhibition by nonsteroidal anti-inflammatory agents of the physiological effects of PGE1 in animals may occur, at least in part, at the level of adenylate cyclase. The possibility that multiple classes of adenylate cyclase-linked PGE receptors might exist in nature is discussed.

N 6, O2'-dibutyryl cyclic AMP (DBcAMP) (3 x 10-4 M) causes reproducible changes in the morphology, growth rate and terminal cell density of 3 different types of human tumour cells grown in culture. Human tumour astrocyte lines 1181N1 and EH-118MG show generalized ablation of membrane systems when grown in the presence of this cyclic AMP analogue. This is not consistent with differentiation; it may be a toxic effect but possibly represents the response of these cell types to DBcAMP. These cells also grow more slowly and to lower terminal cell densities as a function of the concentration of DBcAMP. Although the growth kinetics demonstrate a return of these malignant cells to a state of density-dependent growth, micrographic analyses of inter-cellular relationships reveal that the cells still grow in overlapping patterns. Thus as judged by the commonly accepted criteria, DBcAMP does not induce contact inhibition of growth. All observed changes induced by the presence of DBcAMP are rapidly and completely reversed upon its removal from the growth medium. Human neuroblasts (NB1) grown in the presence of DBcAMP show irreversible morphological changes and the rate and extent of cell division is reduced. There is marked hyperplasia of intracytoplasmic microfilaments, which is considered consistent with differentiation.

Dihydrofolate reductase has been purified 2000-fold from guinea pig liver, and 450-fold from guinea pig small intestine. The chromatographic properties, pH optima, and substrate and cofactor requirements of the two enzymes have been compared; the susceptibility of the enzymes to a series of activators and inhibitors also has been determined. No differences were noted between dihydrofolate reductases from the two sources; it is concluded that the unusually high resistance of the guinea pig to folate antagonists cannot be attributed to differences in the properties of small intestine and liver dihydrofolate reductase, or to differences between guinea pig dihydrofolate reductase and the analogous enzyme from other mammalian species.

As a part of the process of agonist-induced desensitization, 1321N1 human astrocytoma cells lose up to 95% of their beta-adrenergic receptors, as detected by 125I-hydroxybenzylpindolol (125IHYP) binding, after 12-24 h of exposure to isoproterenol. In preconfluent cultures the loss of beta-receptors is completely reversible upon removal of isoproterenol, with receptor levels reaching 100% of control levels within 48-72 h. Addition of cycloheximide (5 micrograms/ml) upon removal of agonist does not prevent the recovery of receptors. After an initial 4-h lag, receptors accumulate in the presence of cycloheximide until the same receptor level is reached that was present at the onset of desensitization. Confluent cultures, which have a reduced number of receptors per cell, recover beta-receptors to only 60 to 70% of control levels following removal of isoproterenol. In addition, cycloheximide blocks the recovery of receptors in these cultures. The effects of cycloheximide on the accumulation of receptors during cell growth suggest that receptors are stable in preconfluent cultures and that turnover only occurs later when cultures are confluent. The data also indicate that long term exposure of cells to catecholamine results in a form of the beta-adrenergic receptor that is undetectable by 125IHYP binding but, nonetheless, retains its primary amino acid structure. The undetectable receptors appear to be retained until agonist is removed, whereupon they become detectable by 125IHYP binding with a t1/2 of about 36 h in the presence of cycloheximide.

In conclusion, our data indicate that exposure of 1321N1 astrocytoma cells to catecholamines initiates a series of reactions that decreases cellular responsiveness to catecholamines and eventually results in a loss of functional beta AR from the cell. Whether the series of reactions depicted in Fig. 1 indeed represents a sequential process is not yet known. The molecular nature of the beta AR modifications responsible for uncoupling, internalization, and loss of binding also remains unknown. Nonetheless, these reactions are not restricted to the beta AR/AC system of 1321N1 astrocytoma or C62B glioma cells, since a number of investigators working with a variety of homogeneous cell systems have reported the occurrence of similar phenomena (2,7,8,13-16,19,21,28). Thus, the basic framework of catecholamine-induced modification of the properties of beta AR in astrocytoma cells is likely representative of the general phenomenon of agonist-specific desensitization of the beta AR-linked AC of mammalian cells.

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.

Human astrocytoma cells (1321N1) in culture respond to pharmacological concentrations of prostaglandins and catecholamines with a marked increase in the accumulation of cyclic AMP. However, growth of 1321N1 cells in the presence of low concentrations (0.003 to 0.1 muM) of prostaglandin E1 (PGE1) results in a marked reduction in the responsiveness of the cells-even to concentrations of PGE1 that normally stimulate maximal accumulation of cyclic AMP. Occasionally, a partial reduction in the responsiveness to catecholamines was observed in cells grown in the presence of PGE1. When it occurred this effect could be correlated with an increase in the cyclic AMP-degradation capacity of the cells. This loss of responsiveness to catecholamines could be completely reversed by 1-methyl-3-isobutylxanthine, a potent inhibitor of phosphodiesterase activity in 1321N1 cells. The consistently observed and more profound desensitization to the effects of PGE1 could not be correlated with an increase in cyclic AM-degradative capacity. Accordingly, 1-methyl-3-isobutylxanthine was only minimally effective in reversing desensitization to PGE1. It is concluded that the inability of 1321N1 cells grown in the presence of PGE1 to accumulate cyclic AMP upon subsequent challenge with PGE1 is primarily due to a selective desensitization of the PGE1-activated adenylate cyclase.

Exposure of postconfluent 1321N1 human astrocytoma cells to 1.0 microM isoproterenol for 12-24 hr results in a 90% loss of beta-adrenergic receptors. Upon removal of agonist, recovery of beta-receptors to control levels occurs within 72 hr. The recovery of receptors is completely blocked by cycloheximide [R. C. Doss, J. P. Perkins, and T. K. Harden, J. Biol. Chem. 256:12281-12286 (1981)]. In contrast cycloheximide does not block recovery of beta-receptors after down-regulation in preconfluent cultures. To determine unambiguously if beta-receptor synthesis accounts for the recovery of receptors after down-regulation, post confluent cultures were incubated with isoproterenol and then transferred to agonist-free medium containing either normal or "heavy" (2H, 13C, 15N) amino acids. The rate and extent of beta-receptor recovery were similar in both normal and heavy amino acid-containing medium. When beta-receptors that had recovered in the heavy amino acid-containing medium were labeled with 125I-cyanopindolol, solubilized in Lubrol PX, and subjected to centrifugation on a 5-15% sucrose density gradient, they exhibited an increased mass compared to beta-receptors that recovered in the presence of normal amino acids. These results confirm that the density shift method is a useful approach for the study of beta-receptor synthesis and that new receptor synthesis occurs during recovery of beta-receptors from catecholamine-induced down-regulation in postconfluent cultures.

Human astrocytoma cells (EH118MG) respond to catecholamines and prostaglandins with a marked increase in the rate of formation of cyclic AMP. Treatment of EH118MG cells with cholera toxin (10 to 100 ng/ml) for 45 to 60 min caused an increase in cellular cyclic AMP content (5- to 10-fold over basal). Cholera toxin also decreased the K0.5 for isoproterenol 10- to 50-fold and decreased the K0.5 for prostaglandin E1 (PGE1)30- to 100-fold, while increasing the maximal response to PGE1 by 1.5- to 3-fold. Treatment with cholera toxin did not change the K1 values for beta-adrenergic receptor antagonists such as propranolol, alprenolol, and sotalol. Direct binding studies using [125I]iodohydroxybenzylpindolol indicated no significant changes in the number of beta-receptors or in the kinetics of the interaction of the radioligand with receptors after treatment of cells with the toxin. Competition binding studies with propranolol and sotalol revealed no toxin-induced change in Kd values for these antagonists. Treatment with cholera toxin caused only small decreases (2- to 3-fold) in the Kd values for binding of isoproterenol and norepinephrine. It is concluded that cholera toxin has little direct effect on the binding of agonists or antagonists to beta-receptors, but instead increases the efficiency of coupling of receptor and catalytic moieties of adenylate cyclase.

For the past several years we and our colleagues have been involved in a study of catecholamine-induced desensitization of the β-adrenergic receptor (βAR)-linked adenylate cyclase system. There appears to be a number of processes utilized by cells to reduce responsiveness to catecholamines (see Lefkowitz et al., 1980; Perkins et al., 1982; Harden, 1983; Hertel & Perkins, 1985 for recent reviews). Our studies have focused on a desensitization process that is βAR-specific, that is, it results in changes in the system that eliminate responsiveness only to catecholamines.

Exposure of certain target cells to hormone agonists not only causes the activation of adenylate cyclase and the accumulation of cyclic AMP but sets in motion a complicated series of events designed to down-regulate the system if exposure to the agonist is extended in time. The development of refractoriness has been shown to result from a number of specifiable phenomena, each exhibiting unique kinetic properties. In a number of systems it has been demonstrated that upon exposure of cells to catecholamines there is a rapid uncoupling of β-adrenergic receptors from adenylate cyclase. This rapidly reversible event results in a selective loss of cellular responsiveness to catecholamines. With continued exposure of cells to catecholamine there occurs a slowly reversible loss of β-receptors from the cell. Evidence from several laboratories suggests that these phenomena are associated with changes in the membrane form of β-adrenergic receptors. In addition, it also has been shown that a nonspecific loss of responsiveness of adenylate cyclase can occur during incubation of cells with hormones that elevate cyclic AMP levels. At least in the C 6 rat glioma cell line, this phenomenon appears to occur as the result of a cyclic AMP-induced increase in the synthesis of a protein that confers on adenylate cyclase a reduced capacity to respond to all activators. The most detailed studies of agonist-induced refractoriness have been carried out with homogeneous cell populations maintained in vitro. However, there is widespread evidence for the occurrence of similar adaptive phenomena in vivo.

The characteristics of catecholamine-induced desensitization of the beta-adrenergic receptor-linked adenylate cyclase system is compared in C62B and C6BD12 rat glioma cells and 1321N1 human astrocytoma cells. The process of receptor-specific (or agonist-specific) desensitization is demonstrated in the C62B subline. Thus, upon exposure of C62B cells to catecholamine a rapid decline (t 1/2 = 6-8 min) in isoproterenol-stimulated adenylate cyclase activity occurs with minimal loss in basal or NaF-stimulated activity. With the same time course an uncoupled population of beta-receptors is formed and can be isolated as a low density, particulate subfraction of cell lysates. These early desensitization processes are not blocked by cycloheximide. With extended exposure to catecholamines beta-receptors are lost from detection with a t 1/2 of 150-180 min. This process in C62B cells exhibits the same characteristics observed by others for other sublines of C6 cells (Homburger, et al. J. Biol. Chem. 255: 10436, 1980; and Fishman, et al. Mol. Pharmacol. 20: 310, 1981) and by us for human astrocytoma cells (Su et al. J. Biol. Chem. 255: 7410, 1980). In addition, the existence in C62B cells of a cyclic AMP-induced, cycloheximide-sensitive desensitization process, such as that reported by others (Terasaki, et al. Adv. Cyclic Nuc. Res. 9: 33, 1978) is confirmed. Thus, it appears that C62B cells exhibit all of the characteristics typically attributed to receptor-specific desensitization in addition to the cyclic AMP-mediated heterologous process described previously.

Incubation of human astrocytoma cells (1321N1) with low concentrations of isoproterenol results in a specific loss of responsiveness to catecholamines as evidenced by a decreased accumulation of cAMP in intact cells, a reduction in isoproterenol-stimulated adenylate cyclase activity, and a decrease in beta-adrenergic receptor density, as measured by the specific binding of 125I-hydroxybenzylpindolol. The kinetics of desensitization suggest the involvement of two different reactions. The initial reaction involves a rapid loss of adenylate cyclase activity with little loss of beta-adrenergic receptors. Subsequently, a slower reaction results in the loss of measurable beta-adrenergic receptors. The degree of loss of both parameters was similar after 24 h of desensitization. It is concluded that the loss of beta-adrenergic receptors is an event that occurs as a result of the initial uncoupling of the beta-receptor-linked adenylate cyclase.

The interactions of agonists and antagonists with beta-adrenergic receptors on intact 1321N1 human astrocytoma and C62B rat glioma cells were studied by using the radioligand (-)-[125I]iodopindolol. Competition binding assays were performed at 37 degrees C under equilibrium conditions and in short-time nonequilibrium assays that approximated initial velocity conditions for binding of the radioligand. The theoretical basis and experimental validation of the initial velocity approach for determining binding affinities of rapidly equilibrating ligands are presented. For the agonists isoproterenol and epinephrine, high binding affinities that approximated their apparent affinities for binding in membranes and for increase of cyclic AMP concentrations in intact cells could be demonstrated only in short-time assays; in contrast, much lower affinities were observed with equilibrium (60-min) assays as reported previously for various cell lines. High-affinity binding of isoproterenol to 1321N1 cells also was observed in equilibrium (6-hr) binding assays carried out on ice. These results indicate that in the native state the intact cell beta-adrenergic receptor has a high binding affinity for agonists and suggest that incubation at 37 degrees C in the presence of an agonist converts the receptors to a form with low affinity for agonists.

Incubation of 1321N1 astrocytoma cells for 15 min with 1 microM (-)-isoproterenol resulted in a 50-65% loss of isoproterenol-stimulated adenylate cyclase activity. No decrease occurred in basal adenylate cyclase activity or in the density of beta-adrenergic receptors as assessed by (125I)-hydroxybenzylpindolol binding. Concentration-effect studies indicated that the apparent affinity of isoproterenol for inhibition of (125I)-hydroxbenzylpindolol binding was decreased by approximately 10-fold in membranes prepared from cells that had been exposed to 1 umM isoproterenol for 15 min. In the presence of GTP there was a shift to the right of the concentration-effect curve for isoproterenol in control membranes. GTP had little effect on the apparent affinity of isoproterenol in desensitized membranes. In desensitized cells that were subsequently washed free of catecholamine, the decrement in isoproterenol-stimulated adenylate cyclase activity and the decrease in the capacity of isoproterenol to inhibit 125IHYP binding returned to control levels within 15 min. These data are consistent with the hypothesis that an early event in the process of desensitization in 1321N1 cells involves a reversible uncoupling of beta-adrenergic receptors and adenylate cyclase.