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R Barber

Publications and source records attributed to R Barber.

At least 19 recordsLinked to original sources
PubMed1996

Collisions and encounters in simulations of receptor/GTP-binding protein interactions via simple diffusion.

In two intact cell systems in which GTP-binding protein (G) activity is initiated by the presence of agonist-bound receptors (R), it has been demonstrated that the rate of G activation is influenced by the rate of turnover of agonist occupancy among the receptor population. G activity is reduced when a low concentration of agonist-occupied receptors comprised by low fractional occupancy of a large receptor population is replaced by the presence of the same concentration of 100%-occupied receptors. This effect has been proposed to be due to a time interval of interaction between R and G (an encounter) that is long compared to the time of a single collection between R and G and long compared to the lifetime of an agonist-receptor complex. In a recent simulation study of R-G interaction via diffusion, the effect of agonist occupancy turnover was observed but it was assumed that long encounters were not operative. In this study, encounter intervals in simulations of R-G interaction by simple diffusion were measured in order to address that difference. The results demonstrate that relatively long encounters comprised of multiple, separate collisions are an inherent part of R-G interaction as modelled by diffusion. The implications for further implementation of simulation studies of R-G interaction are discussed.

Diffusion↗
PubMed1996

Psychiatrists working in primary care: a survey of general practitioners' attitudes.

OBJECTIVES: To collect information on current working arrangements between general practitioners (GPs) and mental health professionals and to assess GPs' attitudes towards developing closer working practices with psychiatrists in the primary care setting. METHOD: Six hundred and three GPs from South Australia were surveyed with questionnaires. Main outcome measures included information about existing primary care links between GPs and mental health professionals, GPs' preferred working arrangements with psychiatrists in the primary care setting and their attitude towards developing these practices, including perceived obstacles, advantages and disadvantages. RESULTS: One hundred and eighty-one completed questionnaires were returned. One in 11 GPs returning the questionnaire (RGPs) had established primary care links with a psychiatrist, 1 in 6 with clinical psychologists and 1 in 17 with psychiatric nurses and social workers. RGPs held positive attitudes towards developing closer links at their work settings with psychiatrists when it le8ads to improved collaboration and access to psychiatrists. Reservations were expressed about the public weakening of the GPs' primary care role. CONCLUSIONS: The joint needs of clinical care and GPs' further training in psychiatry could be addressed by further development of schemes to attract psychiatrists to work in primary care settings. This is mostly viewed very positively by GPs, although the percentage of GPs responding make these conclusions tentative. It is more likely to occur with changes to current funding of both private psychiatric care and GP remuneration, with a recognition of time spent in liaison.

Adult↗
PubMed1996

Stable activation and desensitization of beta 2-adrenergic receptor stimulation of adenylyl cyclase by salmeterol: evidence for quasi-irreversible binding to an exosite.

The relaxation of tracheal smooth muscle by the beta 2-adrenergic receptor (beta AR) agonist salmeterol displays several unusual properties: (i) slow onset of action (t1/2 = 5-15 min), (ii) prolonged activation (t1/2 = 8-14 hr), and (iii) the ability to recover from beta AR blockade. These properties led to the hypothesis that salmeterol binds with very high affinity to an exosite in addition to the beta AR activating site. Despite extensive characterization of salmeterol-induced bronchodilation, little is known about the molecular actions of salmeterol. We report the unique properties of salmeterol binding to the beta AR, activation of adenylyl cyclase, and desensitization of the hamster beta AR expressed in L cells. First, we found that salmeterol activation of adenylyl cyclase, although rapid and potent (low EC50 relative to epinephrine), was nevertheless remarkably inefficient relative to the full agonist epinephrine. Reduced coupling efficiency of salmeterol was demonstrated using formulations recently introduced by our group. Second, we found that pretreatment of L cells with salmeterol led to a stable activation of adenylyl cyclase that survives extensive wash procedures and sucrose step gradient purification of plasma membrane fractions. This activation of basal adenylyl cyclase did not require salmeterol binding to the classic active site during pretreatment, as it occurred in the presence of an excess of a beta AR antagonist. Third, we found that the rapid phase of salmeterol-induced desensitization was much reduced relative to epinephrine, consistent with its poor coupling efficiency and with its prolonged activation of adenylyl cyclase. These unique properties of salmeterol support the proposal that it binds reversibly to the activating or active site and as well to an extremely high affinity exosite from which it has access to the active site.

Adenylyl Cyclases↗
PubMed1996

Repetitive endocytosis and recycling of the beta 2-adrenergic receptor during agonist-induced steady state redistribution.

The human beta 2-adrenergic receptor (beta 2AR) rapidly internalizes after binding agonist, resulting in a dramatic redistribution of receptors from the plasma membrane and into endocytic vesicles. We sought to determine whether intracellular receptors constitute a static pool or represent a fraction of dynamically internalizing and recycling receptors. Using cells expressing a beta 2AR with an epitope tag at its amino-terminal ectodomain, changes in surface receptor levels were measured by flow cytometry and radioligand binding assays. The addition of a saturating level of a strong agonist (isoproterenol) caused the endocytosis of receptors with first-order kinetics (ke for naive cells, 0.222 min-1). After 10 min, the level of surface receptors remained stable at approximately 20% that of untreated cells, even though endocytosis continued with similar kinetics (ke for pretreated cells, 0.258 min-1), suggesting that internalized receptors were cycling in steady state with surface receptors. This prediction was confirmed directly by showing that internalized beta 2ARs recycled to the cell surface in the continued presence of agonist. The calculated transit times (1/k) in the presence of isoproterenol were 3.9 min for endocytosis and 11.2 min for recycling. The endocytic rate constant and the steady state redistribution to the internal pool were much lower after treatment with the partial agonist albuterol, suggesting a correlation between the efficiency of endocytosis and that of receptor coupling to the downstream signal transduction pathway. These findings indicate that in the presence of agonist, beta 2ARs are in a dynamic steady state between the plasma membrane and endosomes that is regulated principally by agonist efficacy.

Adrenergic beta-Agonists↗
PubMed1995

Frontotemporal dementia and Alzheimer's disease: retrospective differentiation using information from informants.

The study examined the feasibility of differentiating frontotemporal dementia from Alzheimer's disease on the basis of retrospective historical information obtained from relatives of patients. A structured questionnaire was devised of patients' symptoms, with emphasis on those cognitive and neuropsychiatric features found in earlier prospective clinical studies to distinguish the two conditions. The questionnaire was given to close relatives of deceased patients in whom the diagnosis of non-Alzheimer's frontotemporal degeneration of Alzheimer's disease had been verified at necropsy. The interviewer had no previous contact or knowledge of those patients, nor clinical experience of patients with frontotemporal dementia. The questionnaire elicited a distinct profile of responses for the two diagnostic groups with emphasis on early personality change, unconcern, and socially inappropriate behaviour in frontotemporal dementia and disturbance in memory and topographical orientation prominent in patients with Alzheimer's disease. A scoring system separated out individual patients with frontotemporal dementia from those with Alzheimer's disease. It is concluded that it is possible to obtain useful information about the precise pattern of dementia from informants even many years after the patient's death. The questionnaire provides the foundation of a diagnostic instrument for use in family history studies of dementia.

Adult↗
PubMed1994

Growth of S49 wild type cells in 3 nM epinephrine increases cyclic AMP phosphodiesterase activity.

In this communication we report that intact cell measurements of cAMP decay have shown an increase averaging over 80% in the cAMP decay constant (kdy) in intact S49 WT cells following a 24 hour growth in 3 nM epinephrine (24 hr/3 nM epinephrine). A comparable percentage increase was seen in cell free PDE activities from cells similarly treated. The enhanced PDE activity and kdy were detectable after 6 hours of incubation with 3 nM epinephrine, and were maximal by 24 hours of incubation. Lysate PDE activity returned to control levels within 24 hours after the cells were transferred to epinephrine-free growth medium. The increased PDE activity present in S49 WT cells after 24 hr/3 nM epinephrine was the result of increased concentrations of the enzymes already present or the expression of very similar enzymes, rather than the expression of enzymes with markedly different characteristics. Firstly, the apparent Km values for the PDE's in lysates from control and 24 hr/3 nM epinephrine cells were both in the region of 1 microM. Secondly, PDE activities in lysates from 24 hr/control and 24 hr/3 nM epinephrine S49 WT cells showed similar sensitivities to PDE inhibitors. There was support for the hypothesis that the increase in PDE activity in 24 hr/3 nM epinephrine S49 WT cells is dependent on the presence of cAPK. That is, although 24 hr/3nM epinephrine caused a distinct homologous desensitization of adenylate cyclase in S49 kin cells, neither kdy nor lysate PDE activity were affected. Additionally, 24 hours incubation of S49 WT cells with 3 microM dibutyryl cAMP resulted in increased PDE activity. Finally, the stimulatory effect of 24 hr/3 nM epinephrine on PDE activity was inhibited in the presence of cycloheximide, suggesting the involvement of protein synthesis in the process. This study shows that prolonged treatment with very low concentrations of epinephrine results in an increase in PDE activity which had a significant effect on cAMP accumulation. The increases were quantified by intact cell and cell free measurements, with kinetic data which were consistent with the hypothesis that the increased PDE activity in 24 hr/3 nM epinephrine cells reflected an increase PDE synthesis which was dependent on activation of the cAPK.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1994

Differential expression of the beta-adrenergic receptor modifies agonist stimulation of adenylyl cyclase: a quantitative evaluation.

The effect of varying levels of beta 2-adrenergic receptor (beta AR) expression on the capacity of the receptor to activate adenylyl cyclase through regulatory G proteins has been systematically explored in this paper, using differential expression of hamster and human beta AR in L cells. Equations have been developed based on the cycle of G protein activation first proposed by Cassel and Selinger and the mobile receptor model, which assumes that hormone-bound beta AR can stimulate a number of different adenylyl cyclase moieties through the G protein during a single cycle of activation. These equations predict the relationship of receptor number to the EC50 (potency) and Vmax (efficacy) for adenylyl cyclase activation. L cell clones were selected with stable expression of the beta AR over a 2000-fold range of levels (from 5 to 10,000 fmol/mg of membrane protein). Experimentally determined values for the EC50 and the Vmax for epinephrine stimulation of adenylyl cyclase over the entire range of receptor levels were found to be in excellent agreement with predictions of the traditionally accepted models. A method is introduced that allows calculation of beta AR coupling efficiency while taking into account the effect of variable receptor levels. The approach provides a quantitative means for the determination of coupling efficiency of the receptor/G protein/adenylyl cyclase system over wide variations in receptor levels and allows for a rational comparison of coupling efficiencies of wild-type and mutant receptors when receptor levels differ.

Adenylyl Cyclases↗
PubMed1993

Analysis of receptor-mediated activation of GTP-binding protein/adenylate cyclase using the encounter coupling model.

The rate of adenylate cyclase activation via agonist-bound receptors in intact cells can be partly dependent on the rate of turnover of occupancy by agonist with respect to individual receptors. For instance, low occupancy of the full complement of receptors by epinephrine in intact S49 cells has been shown to promote a rate of activation that is substantially greater than that for high occupancy of a small number of receptors for which the concentration of epinephrine-bound receptors is the same. According to the encounter coupling model, a partial dependence of the relationship between receptor occupancy and adenylate cyclase activity on the agonist binding frequency can in principle be explained by episodic interactions of finite duration (encounters) between individual pairs of receptor and GTP-binding protein. The mean lifetime of the agonist-receptor complex and the frequency of binding relative to the mean duration of such encounters dictate whether there is variation of the state of the receptor during an encounter and the extent to which the overall rate of GTP-binding protein activation can be dependent on binding frequency. We present here a quantitative analysis of agonist concentration versus cyclase response curves in terms of the encounter coupling model that explicitly includes agonist binding frequency, the encounter frequency, and the encounter duration as parameters. The essential result is that the model is quantitatively consistent with concentration versus response curves for receptor-mediated activation of adenylate cyclase in S49 cells. It is also shown that the model is consistent with data on the differential effects of antagonists to inhibit agonist-stimulated cyclase activation in a manner that is dependent on the antagonist binding frequency.

Adenylyl Cyclases↗
PubMed1992

The encounter coupling model for beta-adrenergic receptor/GTP-binding protein interaction in the S49 cell. Calculation of the encounter frequency.

Experiments measuring epinephrine stimulation of the S49 cell have demonstrated that the rate of adenylate cyclase activation is partly dependent on the rate of turnover of epinephrine occupancy with respect to individual receptors. Specifically, it has been shown that a low occupancy of the full receptor population by epinephrine promotes a rate of adenylate cyclase activation significantly greater than that for a low number of receptors completely occupied by epinephrine with which the concentration of bound receptors is the same. This finding indicated that the interaction of individual receptors with GTP-binding protein (G) occurs on a time scale which is greater than the mean lifetime of the epinephrine-receptor complex; during this period of interaction (an "encounter"), a receptor can change its occupancy state in the presence of a high binding frequency agonist such as epinephrine. Here we present a general analysis, in an extension of the Collision Coupling Model of Tolkovsky and Levitzki (Biochemistry 17: 3795-3810, 1978), of the consequences of encounters (rather than pure collisions) for the relationships of receptor occupancy, receptor-agonist complex lifetime, and receptor-agonist efficiency to G/adenylate cyclase activation. Using this "encounter coupling" model of receptor/G interaction, it is demonstrated from a theoretical standpoint that the net rate of G activation can depend in part on the agonist binding frequency. The predicted dependence is consistent with the data on which the model is based, in which high binding frequency increases the activation rate. A special case of the "encounter coupling model" allows calculation of the frequency of encounters by an analysis of a previous experiment using epinephrine in which the rate of adenylate cyclase activation was measured in response to a small number of fully occupied, highly efficient receptors. Using those results and the model developed here, the encounter frequency was found to be on the order of 100/min in the intact S49 cell. This calculation relied on knowledge of the rate of inactivation of G/adenylate cyclase in intact cells. A method for the measurement of the adenylate cyclase inactivation rate is presented. Using this method, the adenylate cyclase inactivation rate constant was found to be between 0.8 and 3.0/min.

Adenylyl Cyclases↗
PubMed1992

Beta-adrenergic receptor levels and function after growth of S49 lymphoma cells in low concentrations of epinephrine.

Growth of S49 wild-type (WT) lymphoma cells for 24 hr with 3 nM epinephrine produces a very pronounced attenuation of cAMP accumulation in response to subsequent challenges with much higher concentrations of the catecholamine [Mol. Pharmacol. 36:459-464 (1989)]. We report here the effects of this treatment, in S49 WT, cyc-, and kin- cells, on the responsiveness of adenylate cyclase in partially purified membranes. The desensitization of adenylate cyclase in the S49 WT cells after 24-hr treatment was homologous, in that only responses to epinephrine were attenuated. The EC50 for epinephrine stimulation of adenylate cyclase was 54 +/- 8% (mean +/- standard error) higher in treated cells than in controls, and there was a 32 +/- 3% reduction in Vmax at supramaximal epinephrine concentrations. The treatment also caused a 34 +/- 9% reduction in the levels of the beta-adrenergic receptor (beta AR), which was of a sufficient magnitude to account for the homologous desensitization seen. The 24-hr treatment had similar effects in S49 kin- cells, where we observed a 28 +/- 4% decrease in Vmax, a 35 +/- 6% increase in EC50 for epinephrine stimulation of adenylate cyclase, and a 25 +/- 3% decrease in beta AR. In contrast, the 24-hr treatment had no measurable effect on adenylate cyclase activity in S49 cyc- cells. That is, the responsivity of adenylate cyclase reconstituted with Gs from S49 WT cells was not attenuated, although beta AR levels were significantly decreased. The desensitization of S49 cells with the 24-hr treatment was additive with that mediated by the cAMP-dependent protein kinase (cAPK). Further, unlike the cAPK-mediated attenuation, it was relatively insensitive to the levels of free Mg2+ in the adenylate cyclase reaction mixture. The characteristics of the desensitization produced by 24-hr treatment with 3 nM epinephrine, together with the observation that it is similar in S49 WT and kin- cells, demonstrates that the process in WT cells is, at least in part, independent of the rapid cAPK-mediated desensitization. It is also most likely that it is unrelated to the rapid cAMP-independent processes involving sequestration/internalization or the beta AR kinase, because those mechanisms require much higher receptor occupancies than the 0.2% occurring with 3 nM epinephrine. Thus, 24-hr treatment appears to produce attenuation of adenylate cyclase by causing down-regulation of beta AR, without involving any other known form of desensitization.

Adenylyl Cyclases↗
PubMed1992

Positively cooperative cAMP phosphodiesterase attenuates cellular cAMP responses.

We have shown that growth of S49 WT mouse lymphoma cells for 24 hr in 3 nM epinephrine produced very significant desensitization of subsequent cellular cAMP responses to challenges with higher concentrations of epinephrine. The effects of this long-term treatment (LTT) were obvious in intact cells and also when adenylate cyclase activity was measured in semi-purified membranes. Beta 2-adrenergic receptors (beta 2AR) were decreased by LTT, and the desensitization of adenylate cyclase was due at least in part to this down-regulation. When mouse L cells transfected with WT beta 2AR from hamster lung (L-WT beta 2AR cells) were subjected to LTT, the attenuation of adenylate cyclase in membranes was obvious, but the consequences of LTT on intact L-WT beta 2AR cells were highly equivocal. That is, when the effects of epinephrine on cellular cAMP levels were measured in LTT or control L-WT beta 2AR cells little desensitization was apparent. Further, cellular cAMP excursions in response to even very high concentrations of epinephrine were very small in control L-WT beta 2AR cells as compared to control S49 WT cells. Subsequent experiments have shown that L-WT beta 2AR cells possess a phosphodiesterase (PDE) which demonstrates marked positive cooperativity with cAMP with a Hill coefficient of 2. The EC50 for cAMP hydrolysis was approximately 30 nM in cell free preparations. cGMP was a positive allosteric effector of the L-WT beta 2AR cell PDE. Further, when cellular cAMP levels in intact L-WT beta 2AR cells were raised above a threshold by treatment with 0.5 microM forskolin and 2 mM IBMX with the epinephrine challenge, the effect of LLT became obvious in the intact cell system. These data demonstrate that cAMP responses to hormones are greatly decreased in systems where the predominant PDE demonstrates positive cooperativity for cAMP.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Estimation of the kinetic constants for binding of epinephrine to beta-adrenergic receptors of the S49 cell.

The dissociation constant (Kd) for the binding of epinephrine to beta-adrenergic receptors of the S49 cell is 2 microM, which is the ratio of the rate constants for dissociation (koff) and association (kon), Kd = koff/kon. Although the Kd is known by direct measurement, the individual rate constants kon and koff are unknown since they are both too large to be measured by conventional experimental methods. We present here an analysis in which a minimum value for these constants is calculated. The analysis uses a "transiently private receptor" model for coupling of receptors to G protein/adenylate cyclase that is based on the limits prescribed by the known empirical relationship between the beta-adrenergic receptor occupancy by epinephrine (characterized by Kd) and their coupling to adenylate cyclase (characterized by EC50 = 10 nM) as a function of epinephrine concentration. The model makes only the assumption (based on previous evidence) that a receptor cannot activate more than one cyclase during the course of one cycle of binding and unbinding of an epinephrine molecule. In such a model, and with such a large separation between the Kd and the EC50, the rate of G protein/adenylate cyclase activation by epinephrine-bound receptors can be related to the frequency of receptor binding at low concentrations of epinephrine, from which minimum values for the rate constant for association can be derived. This gives the estimates kon greater than 10(8)/M/min and koff greater than 280/min at 37 degrees. The on-rate constant is comparable to the on-rate constants that have been measured for other beta-adrenergic receptor ligands.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1991

Comparisons of the combined contributions of agonist binding frequency and intrinsic efficiency to receptor-mediated activation of adenylate cyclase.

A given overall level of beta-adrenergic receptor occupancy by agonist can involve either high or low turnover of occupancy with respect to individual receptors, depending on the binding properties of the particular agonist. It was recently demonstrated that, for epinephrine-stimulated adenylate cyclase activation in the S49 cell, a portion of the separation between the beta-adrenergic receptor binding curve and the cyclase response curves is dependent on high occupancy turnover (high binding frequency). By involving a larger number of receptors within a short period of time than are bound at any one instant, the effect of high binding frequency is to increase the rate of GTP-binding protein/adenylate cyclase activation over the rate that is observed when the mobility of the number of receptors occupied at any given instant is the rate-limiting factor. This phenomenon contributes to the normal dose-response curve for epinephrine, according to our analysis, but only in combination with the apparent high efficiency of the receptor in the epinephrine-bound state at cyclase activation. Here we examined the potential combination of the contributions of agonist binding frequency and intrinsic efficiency to the adenylate cyclase activation rate for four other beta-adrenergic agonists (isoproterenol, zinterol, metaproterenol, and dobutamine). This was done by a comparison of the response (1-min cAMP accumulation) between a point on the normal dose versus response curve (control) with the response under conditions in which the concentration of agonist-bound receptors was identical to control but the absolute number of receptors involved in maintaining that concentration was significantly reduced. In the experiments, the majority of the receptors were blocked by the beta-adrenergic antagonist propranolol, which has a relatively long occupancy half-life. The remaining receptors were occupied by agonist such that the concentration of bound receptors was identical to the control condition of low occupancy of the full complement of receptors in the absence of antagonist. Compared with control, the experimental condition was one in which agonist occupancy turnover was inhibited and the potential contribution of agonist binding frequency as a factor contributing to the cyclase activation rate was greatly reduced (producing a point on the receptor mobility-limited dose versus response curve). Isoproterenol and metaproterenol show evidence that their binding frequencies and the efficiency of the receptor when bound to them are of such a combination that the normal dose-response curves for these agonists contain a component that is dependent on the binding frequency.(ABSTRACT TRUNCATED AT 400 WORDS)

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1990

cAMP-dependent protein kinase responses of S49 cells are reduced by growth in low epinephrine concentrations.

S49 wild-type mouse lymphoma cells grown in 3 nM epinephrine are extensively desensitized. Cellular cAMP responses to subsequent challenges with 100 nM epinephrine are reduced by as much as 80-90%. In this report, we document that protein kinase activity ratios were also attenuated. For example, the activity ratios in naive cells were increased from 0.26 +/- 0.02 to 0.72 +/- 0.04 by incubation with 100 nM epinephrine for 2 min, whereas in cells grown in 3 nM epinephrine for 24 hr before the experiment they were 0.19 +/- 0.02 and 0.29 +/- 0.03. Attenuated protein kinase activity ratios were obvious at epinephrine challenge concentrations ranging from 10 to 1000 nM. Three kinds of experiments provided evidence that the reduced ratios in desensitized cells were secondary to diminished cAMP responses rather than to changes in the cAMP-dependent protein kinase itself. Firstly, when protein kinase activity ratios were plotted against cAMP levels in naive and desensitized cells, the points fell along a common line. Secondly, cell-free cAMP-dependent protein kinase preparations from naive or epinephrine-treated cells had similar activities in the presence of maximal exogenous cAMP and similar half-maximal cAMP concentrations. Finally, the levels of cAMP-binding proteins in extracts from naive and desensitized cells were essentially identical. We conclude that desensitization of S49 cells by very low levels of epinephrine significantly reduced cAMP-dependent protein kinase responses to much higher concentrations of the catecholamine.

Animals↗
PubMed1989

Evidence for the role of epinephrine binding frequency in activation of adenylate cyclase.

The binding of epinephrine to beta-adrenergic receptors is a rapid-on, rapid-off process, such that at any level of receptor occupancy (defined as the fraction of time a receptor is bound or, alternatively, the probability that any particular receptor is bound at any given instant) the entire population of available receptors has periods of occupancy that occur at high frequency. While in the bound state, the receptor acts as a mobile catalyst for the activation of adenylate cyclase. Two processes, then, could conceivably contribute to the access of epinephrine-bound receptors to cyclase and the extent of cyclase activation for a given concentration of epinephrine: 1) the rapid switching of epinephrine among receptors ensures that discontinuous distributed regions of the cell surface experience agonist activity and 2) the mobility of the receptors (and GTP-binding protein) in the cell membrane makes it possible for one receptor to activate numerous GTP-binding protein-adenylate cyclase complexes. In principle, either effect can lead to a wide separation between the binding and response curves (EC50 much less than Kd). It has so far been assumed that mobility is able to account completely for the separation. The extent of the contribution of the process of agonist binding and unbinding to adenylate cyclase activation has not been demonstrated or quantified. Here we examine the distinction between binding frequency and receptor mobility contributions to adenylate cyclase activation in epinephrine-stimulated S49 lymphoma cells for which there is a 200-fold separation between the EC50 and Kd at 37 degrees (EC50 = 10 nM, Kd = 2 microM). Experiments were designed to measure adenylate cyclase activation rates for a constant concentration of epinephrine-bound receptors but with variation of the absolute number of receptors involved in the activation. This was accomplished by blocking a portion of the receptor population with an antagonist (propranolol) that has a long occupancy half-life, while increasing the occupancy of the remaining receptors by compensating increases in epinephrine. With this protocol, a condition is approached in which receptor mobility alone is responsible for activation. This resulted in a 50% decrease in adenylate cyclase activity, compared with a control of 30 nM epinephrine. Thus, for epinephrine concentrations near the EC50, the switching of epinephrine among the receptor population is necessary for greater than 50% of the observed activity; it can be shown in conjunction that receptor mobility nonetheless accounts for the majority of the separation between the EC50 and the Kd.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenylyl Cyclases↗
PubMed1989

Growth of S49 cells in low concentrations of beta-adrenergic agonists causes desensitization.

Epinephrine at concentrations approximating circulating levels in resting subjects produced significant desensitization in wild type S49 lymphoma cells after long term treatment. Desensitization by such low levels of catecholamines was measured by examining subsequent responses of the cells to higher agonist concentrations and was quantified by comparing the integral cAMP accumulations with time in naive and epinephrine-treated cells challenged with the higher epinephrine concentrations. The cells were significantly desensitized after 8 hr of treatment with 3 nM epinephrine or 3 nM terbutaline and were essentially maximally refractory after 24 hr. The 3 nM epinephrine treatment resulted in a small right shift of the EC50. Responses to epinephrine were partially restored by incubating desensitized cells for 8 hr or longer in growth medium that was free of epinephrine. The attenuation of cAMP responses was largely specific, in that the decrease in the response to prostaglandin was small and the response to forskolin was unchanged. This, together with small increases in cAMP destruction in cell-free preparations from treated cells, suggested that higher phosphodiesterase activity contributed in a minor way to the desensitization. However, the response of the adenylate cyclase system to epinephrine was dramatically attenuated, and very significant changes in the properties of the beta-adrenergic receptors were also obvious. That is, the number of binding sites for epinephrine was reduced by about 65% while the number of sites for [125I]iodocyanopindolol was unchanged. The affinity for the radioactive ligand was significantly reduced. Wild type S49 cells remained viable after several days of continuous treatment with 3 nM epinephrine or terbutaline but responded to subsequent increases in cellular cAMP levels with the expected growth arrest and cytolysis. Involvement of cAMP-dependent protein kinase in this type of desensitization was suggested by the observation that S49 kincells were not desensitized by long term incubation with 3 nM epinephrine. Further, low concentrations of dibutyryl cAMP mimicked the effect of low level epinephrine treatment. We conclude that circulating levels of epinephrine in intact animals are sufficiently high to cause desensitization in cells with sensitivities to the catecholamines in the same range as that of the S49 lymphoma cell in vitro. We would predict that cells with those characteristics would always be at least partially desensitized in vivo.

3',5'-Cyclic-AMP Phosphodiesterases↗