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The American Society for Pharmacology and Experimental Therapeutics has revised the Instructions to Authors for Drug Metabolism and Disposition, Journal of Pharmacology and Experimental Therapeutics, and Molecular Pharmacology These revisions relate to data analysis (including statistical analysis) and reporting but do not tell investigators how to design and perform their experiments. Their overall focus is on greater granularity in the description of what has been done and found. Key recommendations include the need to differentiate between preplanned, hypothesis-testing, and exploratory experiments or studies; explanations of whether key elements of study design, such as sample size and choice of specific statistical tests, had been specified before any data were obtained or adapted thereafter; and explanations of whether any outliers (data points or entire experiments) were eliminated and when the rules for doing so had been defined. Variability should be described by S.D. or interquartile range, and precision should be described by confidence intervals; S.E. should not be used. P values should be used sparingly; in most cases, reporting differences or ratios (effect sizes) with their confidence intervals will be preferred. Depiction of data in figures should provide as much granularity as possible, e.g., by replacing bar graphs with scatter plots wherever feasible and violin or box-and-whisker plots when not. This editorial explains the revisions and the underlying scientific rationale. We believe that these revised guidelines will lead to a less biased and more transparent reporting of research findings.

The physicist E. Rutherford supposedly said, ‘If your experiment needs statistics, you ought to have done a better experiment2005.’ There is a lot of truth to that statement when you are working in a field with a very high signal to noise ratio. In these fields, statistical analysis may not be necessary. But if you work in a field with a lower signal/noise ratio, or are trying to compare the fits of alternative models that don't differ all that much, you need statistical analyses to properly quantify your confidence in your conclusions. I suspect that one of the reasons that the results reported in many papers cannot be reproduced is that statistical analyses are often done as a quick afterthought, with the goal to sprinkle asterisks on figures and the word ‘significant’ on conclusions. The suggestions I propose in this editorial can all be summarized simply: If you are going to analyze your data using statistical methods, then plan the methods carefully, do the analyses seriously, and report the data, methods and results completely.

Measuring the rate and extent of radioligand binding provides information on the number of binding sites, and their affinity and accessibility of these binding sites for various drugs. This unit explains how to design and analyze such experiments. Curr. Protoc. Neurosci. 52:7.5.1-7.5.65. © 2010 by John Wiley & Sons, Inc. Keywords: binding; radioligand; radioligand binding; Scatchard plot; receptor binding; competitive binding curve; IC50; Kd; Bmax; nonlinear regression; curve fitting; fluorescence

BACKGROUND The relation between the sympathetic nervous system and the immune system has not been fully defined. Recent investigations have suggested an adrenergically driven efflux of specific beta 2-receptor-rich lymphocyte subsets into the circulation with either exercise or infusion of exogenous catecholamines. METHODS AND RESULTS To determine whether acute sympathetic stimulation mediates immunoregulatory cell traffic and function via a beta 2-receptor mechanism, we exercised 20 healthy volunteers before and after 1 week of treatment with either the nonselective beta-antagonist propranolol or the beta 1-selective antagonist metoprolol. Before treatment, exhaustive exercise according to the Bruce protocol led to a marked lymphocytosis. Tsuppressor/cytotoxic (Ts/c) and natural killer cells, subtypes with the largest density of beta-receptors, showed the most pronounced increases after exercise, with less impressive elevations in T(helper) and B cells. With respect to function, exhaustive exercise led to a decrease in concanavalin A-stimulated IL-2 receptor expression and [3H]thymidine incorporation while enhancing natural killer cell activity. One week of propranolol therapy blunted the exercise-induced increases in circulating Ts/c and natural killer subpopulations as well as the previously observed alterations in cellular immune function. Treatment with the beta 1-selective antagonist metoprolol, however, did not impair the influence of exercise on any of the above parameters. CONCLUSIONS Acute sympathetic stimulation by exhaustive exercise leads to selective release of immunoregulatory cells into the circulation with subsequent alterations in cellular immune function, either secondary to subset changes or as a result of direct catecholamine effects on function. These changes are attenuated by propranolol but not metoprolol, suggesting a beta 2-mediated mechanism.

A radioligand is a radioactively labeled drug that can associate with a receptor, transporter, enzyme, or any protein of interest. Measuring the rate and extent of binding provides information on the number of binding sites, and their affinity and accessibility for various drugs. Radioligand binding experiments are easy to perform, and provide useful data in many fields. For example, radioligand binding studies are used to study receptor regulation, investigate receptor localization in different organs or regions using autoradiography, categorize receptor subtypes, and probe mechanisms of receptor signaling. This unit reviews the theory of receptor binding and explains how to analyze experimental data. Since binding data are usually best analyzed using nonlinear regression, this unit also explains the principles of curve fitting with nonlinear regression.

To determine the role of the stimulatory guanine nucleotide-binding protein, Gs, and adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase in the basal metabolism of beta-adrenergic receptors in S49 lymphoma cells, we measured the return of receptor number and function after irreversible blockade of receptors. After inactivation of receptors with the irreversible ligand N8-(bromoacetyl)-N'-[3-(4-indolyoxy)-2-hydroxypropyl]-(2)-1,8-diam ino-p- methane (BIM), beta-adrenergic receptors (defined as [125I]iodocyanopindolol binding sites) reappeared in a biphasic manner, the faster phase having a half-time (t 1/2) of 3-8 h (approximately 50% of the sites) and the slower phase greater than 40 h. Although the slow phase is not readily explained, recovery of binding sites during the first 10 h matched recovery of receptor function after BIM treatment (as measured by stimulation of cAMP accumulation) and recovery of receptor sites after downregulation induced by the agonist isoproterenol. Thus quantifying receptor recovery during the first 10 h after BIM treatment appears to be a reasonable method for examining basal receptor metabolism in S49 cells. Measured in this way, metabolism of beta-adrenergic receptors is very similar in wild-type S49 and the following variant clones: cyc- (absent Gs alpha), UNC and H21a (defective Gs alpha), and kin- (lacking cAMP-dependent protein kinase activity). Although previous data have demonstrated that agonist-promoted downregulation of beta-adrenergic receptors requires functional receptor-Gs coupling, the current data suggest that neither Gs nor cAMP-dependent protein kinase activity plays an important role in the regulation of basal metabolism of beta-adrenergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

1. The effects of increases in intracellular adenosine 3':5'-cyclic monophosphate (cyclic AMP) on mitogen-induced generation of inositol phosphates and increases in intracellular Ca2+ concentration were investigated in human peripheral blood mononuclear leukocytes (MNL). 2. The mitogens concanavalin A (Con A), pokeweed mitogen (PWM) and phytohaemagglutinin (PHA) concentration-dependently stimulated generation of inositol phosphates. Catecholamines inhibited this process with an order of potency: isoprenaline greater than adrenaline greater than noradrenaline indicating involvement of beta 2-adrenoceptors. This order of potency was also consistent with the catecholamine potencies for stimulating the generation of cyclic AMP. 3. In addition to catecholamines, the cyclic AMP formation-stimulating agents prostaglandin E1 (PGE1) and forskolin concentration-dependently inhibited mitogen-induced inositol phosphate generation, too. Moreover, the inhibitory effect of isoprenaline was potentiated by co-incubation with the phosphodiesterase inhibitor isobutylmethylxanthine demonstrating that these inhibitory effects were mediated by cyclic AMP. 4. Con A and PHA concentration-dependently increased the intracellular Ca2+ concentration in human MNL (assessed by the fluorescent indicator dye Fura-2). This increase was almost completely blocked by chelation of extracellular Ca2+, demonstrating influx rather than mobilization from intracellular stores. 5. The elevation of intracellular Ca2+ was not blocked by pretreatment with pertussis toxin, 100 ng ml-1, for 16 h. 6. Isoprenaline, PGE1, and forskolin, however, inhibited the mitogen-stimulated elevation of intracellular Ca2+. This inhibition was enhanced by the phosphodiesterase inhibitors isobutylmethylxanthine and Ro 20-1724, demonstrating mediation by cyclic AMP. 7. We conclude that catecholamines and other cyclic AMP increasing agents can inhibit mitogen-stimulated generation of inositol phosphates and elevation of intracellular Ca2+ in resting human MNL.(ABSTRACT TRUNCATED AT 250 WORDS)

Based on in vitro data, previous investigators have hypothesized that Ca2+ entry blockers might affect lymphocyte activation, proliferation and effector function. We have tested this hypothesis by comparing the in vitro and in vivo effects of the Ca2+ entry blocker verapamil on human lymphocytes. In vitro high concentrations of verapamil (100 microM) inhibited mitogen-stimulated Ca2+ influx, inositol phosphate generation, and proliferation; similar effects were observed with diltiazem and nifedipine. In vivo treatment of healthy volunteers with verapamil (2-4 times 240 mg per day for 7 days) did not affect the number of circulating lymphocytes or their subset distribution. Moreover, we did not observe any effect of in vivo treatment with verapamil on mitogen-stimulated lymphocyte proliferation or expression of interleukin-2 receptors in vitro. We conclude that the inhibitory effects of verapamil on lymphocyte activation in vitro are unlikely to be of therapeutic relevance and may not be related to the Ca2+ entry blocking effects of this drug.

Many types of data are best analyzed by fitting a curve using nonlinear regression, and computer programs that perform these calculations are readily available. Like every scientific technique, however, a nonlinear regression program can produce misleading results when used inappropriately. This article reviews the use of nonlinear regression in a practical and nonmathematical manner to answer the following questions: Why is nonlinear regression superior to linear regression of transformed data? How does nonlinear regression differ from polynomial regression and cubic spline? How do nonlinear regression programs work? What choices must an investigator make before performing nonlinear regression? What do the final results mean? How can two sets of data or two fits to one set of data be compared? What problems can cause the results to be wrong? This review is designed to demystify nonlinear regression so that both its power and its limitations will be appreciated.

1. One hypothesis to account for altered adrenergic response in hypertensive patients is alteration in adrenergic receptors on responsive cells. We therefore used radioligand binding methods to examine the α2-adrenergic receptors on platelets isolated from 17 normal men and from 19 men with essential hypertension. in these studies we used the α2-selective radioligand [3H]yohimbine to determine receptor number and affinity on intact platelets. 2. The median number of receptors per platelet was 265 for the hypertensive patients versus 246 for the platelets of controls. Likewise there was no difference between hypertensives and controls in the dissociation constant of the receptors for [3H]yohimbine or adrenaline. 3. Anti-hypertensive treatment with the α2-agonist guanabenz or the β-antagonist propranolol did not change the number or affinity of platelet α2-receptors. 4. in untreated hypertensives the receptor number did not correlate with age, blood pressure, or 24 h urinary excretion of catecholamines or Na+. 5. We conclude that neither hypertension nor anti-hypertensive treatment alters the number of α2-adrenergic receptors on human platelets. Furthermore, because therapy with an α2-agonist does not alter the receptor number and because the receptor number did not correlate with urinary excretion of catecholamines (an index of sympathetic-nervous-system activity), we conclude that ‘down-regulation’ of human platelet α2-adrenergic receptors may not readily occur in vivo.

We have used radioligand binding techniques and subcellular fractionation to assess whether changes in expression of myocardial alpha 1- and beta-adrenergic receptors are mediated by a redistribution of receptors between various membrane fractions. Three fractions were prepared from the left ventricles of guinea pigs that underwent either 1 h of ischemia or injection of epinephrine (0.25 mg/kg ip): a crude membrane, a purified sarcolemma, and a light vesicle fraction. In control animals alpha 1-adrenergic receptors ([3H]prazosin binding) in light vesicles was only 25% of the total alpha 1-receptor density found in sarcolemmal and light vesicle fractions as compared with 50% for beta-adrenergic receptors ([125I]iodocyanopindolol binding sites). Although ischemia was associated with a 53% decrease in the number of light vesicle beta-adrenergic receptors and a 42% increase in the number of sarcolemma beta-receptors (P less than 0.05), there was no change in the number of light vesicle alpha 1-receptors, even though the number of sarcolemmal alpha 1-receptors increased 34%. Epinephrine treatment promoted internalization of beta-adrenergic receptors; sarcolemma beta-receptors decreased 37% and light vesicle beta-receptors increased 28% (P less than 0.025). For alpha 1-receptors, epinephrine treatment decreased the number of sarcolemmal receptors 41% (P less than 0.025) but failed to increase the number of receptors in the light vesicle fraction. The changes in receptor binding to beta-adrenergic receptors in sarcolemmal fractions were mirrored by parallel changes in isoproterenol-stimulated adenylate cyclase activity. These results indicate that alpha 1- and beta-adrenergic receptors may undergo a different cellular itinerary in guinea pig myocardium.(ABSTRACT TRUNCATED AT 250 WORDS)

Which subtype(s) of the alpha-adrenergic receptor occurs on human platelets? Studies of platelet responsiveness to adrenergic compounds and indirect radioligand binding studies addressing this question have yielded contradictory conclusions. These bindings studies employed the ligand [3H]dihydroergocryptine ( [3H]DHE), an alpha-adrenergic antagonist that does not select between alpha 1- and alpha 2-adrenergic receptors and that also binds to other receptor types in some tissues. To determine the subtype of the platelet alpha-adrenergic receptor, we have examined the binding to intact human platelets of [3H]prazosin (alpha 1-selective), [3H]yohimbine (alpha 2-selective), and [3H]rauwolscine (alpha 2-selective), and we have compared the binding of these selective radioligands with that of [3H]DHE. [3H]Yohimbine and [3H]rauwolscine both bound with high affinity (Kd = 2.7 and 4.6 nM, respectively) to an equal number and a single class (Hill coefficient approximately 1.0) of sites ( approximately 300 per platelet), but [3H]yohimbine yielded lower nonspecific binding than did [3H]rauwolscine. In paired experiments, [3H]DHE bound to 1.5 times as many (phentolamine-displaceable) sites as did [3H]yohimbine or [3H]rauwolscine. Unlabeled yohimbine and epinephrine competed for fewer [3H]DHE binding sites than did phentolamine. Thus, in addition to binding to the alpha 2-adrenergic receptors identified by [3H]yohimbine and [3H]rauwolscine, [3H]DHE seems to bind to other sites on human platelets. The nature of these sites is not clear. We found that [3H]prazosin did not identify alpha 1-adrenergic receptors on platelets, and that phenoxybenzamine only inhibited [3H]yohimbine and [3H]DHE binding at higher concentrations than usually observed for alpha 1-adrenergic receptors. We conclude that (1) all alpha-adrenergic sites on human platelets are of the alpha 2 subtype, (2) [3H]DHE may bind to additional, as yet ill-defined, sites in addition to those sites identified by [3H]yohimbine and [3H]rauwolscine, and (3) [3H]yohimbine is the preferred antagonist radioligand for studying the alpha 2-adrenergic receptors on human platelets.

We have examined several features of the regulation of cyclic AMP accumulation in lymphoid cells isolated from peripheral blood of human subjects and in the murine T-lymphoma cell line, S49, S49 cells are unique because of the availability of variant clones with lesions in the pathway of cyclic AMP generation and response. We found that human lymphoid cells prepared at 4 degrees C showed substantially greater cyclic AMP accumulation in response to histamine and the beta-adrenergic agonist isoproterenol than did cells prepared at ambient temperature. The muscarinic cholinergic agonist carbamylcholine and peptide hormone somatostatin failed to inhibit cyclic AMP accumulation in human lymphoid cells and treatment with pertussis toxin (which blocks function of Gi, the guanine nucleotide binding protein that mediates inhibition of adenylate cyclase) only minimally increased cyclic AMP levels in these cells. Thus the Gi component of adenylate cyclase appears to play only a small role in modulating cyclic AMP levels in this mixed population of lymphoid cells. Incubation of whole blood with isoproterenol desensitized human lymphocytes to subsequent stimulation with beta agonist. This desensitization was associated with a redistribution of beta-adrenergic receptors such that a substantial portion of the receptors in intact cells could no longer bind a hydrophilic antagonist. Wild-type S49 lymphoma cells showed a similar redistribution of beta-adrenergic receptors after a few minutes' incubation with agonist. Based on studies in S49 variants, this redistribution is independent of components distal to receptors in the adenylate cyclase/cyclic AMP pathway. By contrast, a more slowly developing, agonist-mediated down-regulation of beta-adrenergic receptors was blunted in variants with defective interaction between receptors and Gs, the guanine nucleotide binding protein that mediates stimulation of adenylate cyclase. Unlike results in human lymphoid cells, S49 cells show a prominent inhibition of cyclic AMP accumulation mediated by Gi; this inhibition is promoted by somatostatin and blocked by pertussis toxin. Inhibition by Gi is unable to account for the marked decrease in ability of the diterpene forskolin to maximally stimulate adenylate cyclase in S49 variants having defective Gs. These results emphasize that both Gs and Gi component are important in modulating cyclic AMP accumulation and receptors linked to adenylate cyclase in S49 lymphoma cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Purified sarcolemmal and light vesicle (intracellular) fractions of beta-adrenergic receptors were used to examine the effects of propranolol on receptor translocation in guinea pig heart. Guinea pigs were given propranolol (0.15 mg/kg/hr) via minipumps for 7 days and either killed or made ischemic for 1 hour via a coronary ligature. Propranolol treatment led to an externalization of beta-receptors from light vesicle to sarcolemmal fractions. This externalization increased the number of surface beta-adrenergic receptors that were functional, as assessed by isoproterenol-stimulated adenylate cyclase activity. After chronic propranolol treatment, ischemia did not further alter receptor distribution. These results suggest that externalization of beta-adrenergic receptors from a light vesicle fraction to the sarcolemma contributes to up-regulation of beta-receptors that occur in response to both propranolol treatment and ischemia. Because propranolol-treated animals show blunting in externalization after myocardial ischemia, propranolol treatment and myocardial ischemia appear to access the same pool of intracellular beta-adrenergic receptors. Depletion of this pool of receptors along with receptor blockade may thus contribute to the mechanism by which the drug is efficacious in preventing some adverse effects of ischemia.

Modulation of neutrophil activation by catecholamines may reflect regulatory mechanisms that couple beta-adrenergic and N-formyl peptide receptors to antagonistic biochemical pathways. We examined kinetic and mechanistic aspects of the inhibition by catecholamines of neutrophil activation by formyl peptides. Inhibition of oxidant production by isoproterenol (ISO) was detected as low as 3 nM, had an ID50 of 10−7 M, and could be blocked and reversed by propranolol. Recovery of cell function occurred over a period of minutes when the concentration of ISO was less than 10−6 M. These observations are discussed in terms of the interaction of ISO with the adrenergic receptors. The site of catecholamine action is addressed. ISO neither influences formyl peptide-receptor interaction nor does it inhibit oxidant production by phorbol ester. These results suggest an impairment of intracellular signalling processes that couple the formyl peptide-receptor binding to cell activation. We observed inhibition of intracellular Ca+ + elevation by ISO only at low formyl peptide concentrations. This inhibition is consistent with a partial inhibition of phosphoinositide metabolism, which was observed. Several other cell responses, including actin polymerization and right angle light scatter, are minimally inhibited by 10−6 M ISO indicating that the cell activation process is not entirely obliterated. The presence of catecholamine and formyl peptide results in a synergistic elevation of cAMP. The intracellular targets of ISO action may be regulated by cAMP dependent kinases and could follow a branchpoint in the activation sequence that leads distinctly to oxidase activation and cytoskeletal activation.

Epinephrine causes platelet aggregation and secretion by interacting with alpha 2-adrenergic receptors on the platelet surface. Platelet aggregation requires the binding of fibrinogen to a specific receptor on the membrane glycoprotein IIb-IIIa complex. Although the IIb-IIIa complex is identifiable on the surface of resting platelets, the fibrinogen receptor is expressed only after platelet activation. The current studies were designed to examine the effect of occupancy of platelet alpha 2-adrenergic receptors by epinephrine on the expression of fibrinogen receptors and on the aggregation of platelets. The ability of epinephrine to induce the expression of fibrinogen receptors was studied under two different conditions: acute stimulation (less than 1 min) and prolonged stimulation (50 to 90 min), the latter of which is associated with a reduction or “desensitization” of the platelet aggregation response. Expression of the fibrinogen receptor was monitored with 125I-fibrinogen as well as with 125I-PAC-1 (PAC-1), a monoclonal antibody that binds to the glycoprotein IIb-IIIa complex only after platelets are activated. Epinephrine caused an immediate increase in PAC-1 and fibrinogen binding that was dependent on occupancy of the alpha 2-receptor by epinephrine and on the presence of extracellular free Ca (KCa = 30 mumol/L). By itself, 1 mmol/L Mg was unable to support induction of the fibrinogen receptor by epinephrine. However, it did decrease the Ca requirement by about two orders of magnitude. Prolonged stimulation of unstirred platelets by epinephrine led to a 70% decrease in the aggregation response when the platelets were subsequently stirred. Despite their decreased aggregation response, desensitized platelets bound PAC-1 and fibrinogen normally, indicating that the loss of aggregation was not due simply to a decrease in fibrinogen receptor expression. Although desensitization was not affected by pretreatment of the platelets with aspirin, it was partially prevented when extracellular Ca was chelated by EDTA during the long incubation with epinephrine. These studies demonstrate that once platelet alpha 2-adrenergic receptors are occupied by epinephrine, extracellular Ca is involved in initiating the aggregation response by supporting the induction of the fibrinogen receptor and the binding of fibrinogen. Furthermore. Ca-dependent reactions subsequent to fibrinogen binding may be necessary for maximal platelet aggregation and are impaired when platelets become desensitized to epinephrine.

Human erythroleukemia cells are a model system for studies of alpha 2-adrenergic receptors and their coupling to inhibition of adenylate cyclase (McKernan, R. M., Howard, M. J., Motulsky, H. J., and Insel, P. A. (1987) Mol. Pharmacol. 32, 258-265). Using Fura-2, we show that alpha 2-adrenergic receptor stimulation also increases intracellular Ca2+ in these cells by 80-250 nM. Although epinephrine only inhibited forskolin-stimulated cAMP generation when beta-adrenergic receptors were blocked, the Ca2+ increase was not affected by beta-adrenergic receptor blockade. The Ca2+ increase was not affected by forskolin or 8-bromo-cAMP. Thus, alpha 2-adrenergic receptors independently couple to elevation of intracellular Ca2+ and adenylate cyclase inhibition. Chelating all extracellular Ca2+ did not reduce the response, demonstrating mobilization of intracellular, rather than influx of extracellular Ca2+. The epinephrine-stimulated Ca2+ mobilization occurred prior to any detectable increase in inositol-(1,4,5)-trisphosphate. It was abolished by pretreatment with pertussis toxin (which blocks some G protein-mediated processes), but not by aspirin and indomethacin (which inhibit cyclooxygenase), nordihydroguaiaretic acid (which inhibits lipoxygenase), or Na+-free buffer (to block any Na+H+ exchange). We conclude, therefore, that alpha 2-adrenergic receptors on human erythroleukemia cells couple to mobilization of intracellular Ca2+ via a (pertussis toxin-sensitive) G protein-mediated mechanism that is independent of inhibition of adenylate cyclase.

Recent studies indicate that antagonism of calcium channels may not be the only mechanism whereby drugs such as verapamil alter myocardial function. We have examined the effect of verapamil on the binding of [3H]prazosin (alpha 1-adrenergic), [3H]quinuclidinyl benzilate (QNB, muscarinic) and [3H]dihydroalprenolol (DHA, beta-adrenergic) to membranes prepared from rat heart. Verapamil competed for the binding of these radioligands in the following rank order: [3H]prazosin greater than [3H]QNB greater than [3H]DHA (Ki for verapamil = 0.6 microM, 7 microM, and 72 microM, respectively). Verapamil (10 microM) competitively inhibited [3H]prazosin binding to rat ventricular membranes; the apparent dissociation constant (KD) of [3H]prazosin increased from 0.13 +/- 0.02 to 1.5 +/- 0.6 nM (SD) without change in maximal binding capacity (Bmax). The effect of verapamil on the affinity of [3H]prazosin was completely reversed by washing the membranes. The verapamil derivative D-600 also inhibited [3H]prazosin binding (Ki = 1.1 microM). Verapamil (30 microM) competitively inhibited [3H]QNB binding in both atria and ventricles and increased the apparent KD of [3H]QNB fivefold (from 0.07 nM to 0.32 nM) without decreasing Bmax. Verapamil was a less potent inhibitor of [3H]DHA binding and its effect was noncompetitive: the KD for DHA was unaltered by 100 microM verapamil while the Bmax decreased severalfold. We conclude that verapamil, at concentrations clinically achieved in the myocardium (approximately 1 microM), competitively inhibits binding to alpha 1-adrenergic and muscarinic receptors and that this inhibition may play a role in the effects of verapamil on the heart.

In animals injected with a bolus of isoproterenol, beta-adrenergic receptors in both mononuclear leukocytes (MNL) and heart were sequestered away from the cell surface, and the time course (0-120 min) and dose-response patterns were similar in the two tissues. In guinea pigs given a constant infusion of isoproterenol, 0.15 mg/(kg.h), down-regulation of total receptor number occurred more quickly and to a greater extent in the MNL than in the heart. We also compared receptor sequestration after aortic constriction-induced acute heart failure. Negligible sequestration (9%) of beta-adrenergic receptors occurred in the MNL of animals treated in this manner, whereas the number of receptors in the sarcolemmal fraction decreased 61%. This selective sequestration of cardiac receptors may result from the action of high concentrations of norepinephrine (which is selective for beta 1 over beta 2 receptors) present at sympathetic nerve-cardiac cell synapses. We conclude that although receptor redistribution occurs similarly in MNL and heart in response to a circulating nonselective agonist, beta-adrenergic receptor redistribution may occur selectively in the heart in response to such stimuli as aortic constriction-induced acute heart failure that activate the sympathetic nervous system.

VERAPAMIL is a calcium-channel blocking agent that has been used extensively in the treatment of coronary vasospasm1 and supraventricular arrhythmias.2 , 3 Its negative inotropic and chronotropic a...

Mononuclear leukocytes are easily accessible cells for investigating the regulation of beta-adrenergic receptors in humans. We have previously shown that brief incubations with agonists redistribute (? internalize) most of the beta-adrenergic receptors on mononuclear leukocytes away from the cell surface without changing total receptor number. However, negligible redistribution occurred after exercise or an infusion of isoproterenol. The current study was designed to ask whether receptor redistribution occurs over a longer time course after administration of terbutaline, a beta 2-adrenergic agonist that is known to cause a decrease in receptor number. Normal volunteers were given terbutaline, 5 mg t.i.d. for 6 days. As expected, the number of beta-adrenergic receptors decreased. However, the remaining receptors were not redistributed. Redistribution also did not occur after 1 or 2 days of terbutaline, at which time down regulation was minimal. We also found that terbutaline did not alter the ability of the receptors to be redistributed or desensitized by a preincubation with isoproterenol.

Studies of beta-adrenergic receptors on several types of intact target cells indicate that agonists, but not antagonists, show prominent KD/Kact discrepancies–lower affinities (KD) in equilibrium binding studies (i.e. in competition with antagonist radioligands) than in functional assays (Kact). In this report we show that intact S49 lymphoma cells initially bind beta-adrenergic agonists in a high affinity manner but that this high affinity binding rapidly converts to a low affinity state. This time course is similar to that of agonist-mediated desensitization in S49 cells. In competitive binding studies conducted with [125I]iodocyanopindolol or [125I]iodohydroxybenzylpindolol, IC50 values for beta-adrenergic agonists increased 13-200-fold between 1 and 60 min, whereas antagonists yielded similar IC50 values at the two time points. The binding of antagonists, but not agonists, could be simulated by a computer model based on the law of mass action. In contrast with results in intact S49 cells, crude membrane fractions yielded similar IC50 values for agonists in 1- and 60-min incubations with [125I]iodocyanopindolol. Moreover, time-dependent decreases in apparent affinity of the agonist (-)-isoproterenol were observed not only in wild type S49 cells but also in several S49 variants (UNC, cyc-, H21a) having defects in Ns, the guanine nucleotide binding protein that couples receptors to activation of adenylate cyclase, and in Kin-, an S49 variant with absent cAMP-dependent protein kinase activity. These results show that beta-adrenergic receptors of intact S49 cells demonstrate a prominent time-dependent decrease in apparent affinity for agonists and that this decrease in affinity does not require cAMP generation, the Ns components defective in several S49 variants or cAMP-dependent protein kinase. The rapid conversion of agonist binding from high to low affinity can account for the rapid desensitization of intact cells to catecholamines and probably explains previously reported KD/Kact discrepancies of intact cells.

Epinephrine-promoted release of [ 3 H]guanylylimidodiphosphate ([ 3 H]Gpp(NH)p) from human platelet membranes has been used to probe the interactions between alpha 2 -adrenergic recpetors and N i , the guanine nucleotide binding protein that couples those receptors to an inhibition of adenylate cyclase activity. We show here that ADP, which also acts through specific platelet receptors to inhibit adenylate cyclase activity, also promotes the release of [ 3 H]Gpp(NH). The amount of [ 3 H]Gpp(NH)-release elicited by epinephrine and by ADP together is equal to the sum of the amounts released by the two agents acting individually. Furthermore the maximal amounts of [ 3 H]Gpp(NH)-release elicited by each of the two agents approximates the numbers of receptors for ADP and epinephrine present in the platelet membranes. These results suggest that the two receptor types interact with distinct portions of the pool of N i molecules and that each receptor initiates guanine-nucleotide exchange on a single molecule of N i .

Radioligand binding experiments are being used to probe a variety of receptor types. Although most such experiments have been conducted with membrane preparations, many investigators are now using radioligand binding techniques to study the interaction of agonists with receptors on intact cells. Often these experiments have been analysed using techniques developed for studies of antagonist binding to membrane fragments. Harvey Motulsky , Lawrence Mahan and Paul Insel wish to point out several flaws in such analyses.

A drug which increases the K d of a radioligand without altering its B max has a competitive interaction with the receptors. Although such data occur most commonly in simple situations in which the drug and radioligand bind reversibly to a common binding site without altering the receptors in other ways, Antonio DeBlasi and Harvey Motulsky point out that similar data can also be obtained in more complicated situations.

Although quinidine is known to have antiadrenergic effects in the cardiovascular system, the precise mechanism by which it exerts these effects is not well defined. We asked whether quinidine binds directly to adrenergic receptors. Radioligand-binding assays were used to identify alpha 1-adrenergic receptors [( 3H]prazosin-binding sites) on membranes prepared from rat heart and kidney, alpha 2-adrenergic receptor [( 3H]yohimbine-binding sites) on human platelets and rat kidney membranes, and beta-adrenergic receptors [( 125I]iodocyanopindolol-binding sites) on rat heart and kidney membranes. Although it did not effectively compete for binding to beta-adrenergic receptors, quinidine competed for binding to alpha 1- and alpha 2-adrenergic receptors and yielded equilibrium dissociation constants of 0.3-3 microM. Two other antiarrhythmic agents, lidocaine and procainamide, did not compete for binding to alpha-adrenergic receptors. Further experiments demonstrated that the interactions of quinidine with the cardiac alpha 1- and platelet alpha 2-adrenergic receptors were competitive and reversible. We conclude that that antiadrenergic actions of quinidine can be explained by occupancy and competitive blockade of alpha 1- and alpha 2-adrenergic receptors.

Saturation experiments using increasing concentrations of radioligand are commonly used to determine receptor number and affinity, but this protocol is not feasible in all situations. Alternatively, competitive binding experiments are often performed in which binding of a single concentration of radioligand is completed for by multiple concentrations of the same unlabelled ligand, but the analysis of such data has been difficult. Antonio DeBlasi and colleagues present here a simple method for calculating receptor number and affinity from competitive binding data. This experimental protocol is useful when a single class of binding site is present, but is often not able to detect the presence of two classes of site.

Summary: The sympathetic co-transmitter neuropeptide Y (NPY) inhibits cardiac contractility in vivo in the rat heart. We tested whether NPY alters the force of contraction or the activity of adenylate cyclase in the isolated human right atrium. Neuropeptide Y did not affect basalor isoproterenol-stimulated force of contraction and did not inhibit forskolin-stimulated adenylate cyclase activity. We conclude that NPY does not directly decrease cardiac contractility in humans. The inhibitory cardiac effects of NPY are likely to be secondary to coronary vasoconstriction, presynaptic inhibition of catecholamine release, and/or baroreflex activity.

We used elution of radioligands at low pH to quantitate intracellular beta-adrenergic receptors on intact S49 lymphoma cells. We validated this method with respect to cell viability, beta-adrenergic receptor integrity, and transferrin receptors on these cells. On control cells, about 15% of the radiolabeled beta-adrenergic antagonists [3H]dihydroalprenolol and [125I]iodocyanopindolol specifically bound at 37 degrees C could not be eluted at low pH; these binding sites appear to be intracellular receptors that are inaccessible to the surface-restricted antagonist [3H]CGP-12177 [tritiated (+/-)-4-(3-t-butylamino-2-hydroxypropoxy)benzimidazole-2-one hydrochloride]. Incubation of cells with the agonist isoproterenol at 37 degrees C for 15 min did not change the number of [3H]dihydroalprenolol binding sites but reduced [3H]CGP-12177 binding sites by 50% or more. However, all specifically bound [3H]CGP-12177 and [3H]dihydroalprenolol were eluted by acid. In addition, the number of acid-elution-resistant [125I]iodocyanopindolol binding sites was not increased in cells coincubated with 1 microM isoproterenol and [125I]iodocyanopindolol for 15 min at 37 degrees C, even though those sites show a loss in apparent affinity for isoproterenol of about 2 orders of magnitude, a loss previously attributed to internalization. We conclude that the early phase of agonist-mediated desensitization of beta-adrenergic receptors in S49 cells does not coincide with the movement of receptors to intracellular sites; instead, agonist-modified receptors remain in association with the plasma membrane and are accessible to the extracellular environment. These "redistributed" receptors together with "cell-surface" and "intracellular" receptors represent three classes of beta-adrenergic receptors that can be selectively identified in intact target cells.

The effects of Na+ on alpha 2-adrenergic receptors coupled to inhibition of adenylate cyclase have been well studied. Effects of Na+ on other receptor types also linked to adenylate cyclase inhibition are not clear. We therefore studied the effects of Na+ on the muscarinic cholinergic receptor of rat ventricular myocardium. Intact ventricular myocytes bound the muscarinic receptor antagonist radioligand [3H]quinuclidinyl benzylate with high affinity (Kd 72 +/- 18 pM) to a large number of sites (138,000 +/- 25,000/cell). In ventricular membranes, Na+ (100 mM) increased receptor affinity for radioligand two-fold and decreased receptor affinity for agonist (carbachol) twofold. Sodium was not required for and did not alter the pattern of cholinergic inhibition of adenylate cyclase in ventricular membranes. We conclude that Na+ has minimal effects on both binding and function of cardiac cholinergic receptors in rat. Our results suggest that monovalent cations are not important regulators of agonist binding and function of cardiac muscarinic cholinergic receptors.

Neuropeptide Y (NPY) appears to be a transmitter of the sympathetic nervous system, and its actions are similar to those of alpha 2-adrenergic receptor stimulation. In human erythroleukemia (HEL) cells, both NPY and epinephrine (acting through alpha 2-adrenergic receptors) inhibit adenylate cyclase and mobilize intracellular Ca++. We investigated possible interactions between NPY and epinephrine. In radioligand binding assays NPY did not alter antagonist or agonist binding to alpha 2-adrenergic receptors. NPY and epinephrine did not act synergistically to elevate intracellular Ca++. Neither agent alone, nor both together, affected the intracellular pH of HEL cells. Preincubation with NPY (like epinephrine) redistributed the alpha 2-adrenergic receptors away from the cell surface and into a sequestered pool.

We studied the regulation of beta-adrenergic receptors in human mononuclear leukocytes (MNL). Total receptor number was determined as specific binding at 4 C of [3H] dihydroalprenolol or [125I]iodopindolol, and redistributed receptors were defined as those binding sites to which the hydrophilic antagonist CGP-12177 did not have access. Receptor function was assessed as cAMP accumulation stimulated by isoproterenol. In in vitro experiments, high concentrations of isoproterenol desensitized receptor function and promoted redistribution of about 80% of the receptors away from the cell surface. However, three in vivo protocols (upright posture for 3 h, moderate exercise, and infusion of isoproterenol for 30 min) redistributed few beta-adrenergic receptors on MNL. The 30-min isoproterenol infusion did not alter later cAMP accumulation, but posture change and exercise increased isoproterenol-stimulated cAMP accumulation in intact MNL. Infusion of isoproterenol for 120 min redistributed 9 +/- 2% (+/- SEM) of the receptors and decreased isoproterenol-stimulated cAMP accumulation by 19 +/- 6%. Isoproterenol-stimulated adenylate cyclase activity in membranes isolated from MNL previously was found to be decreased with upright posture, and we confirmed these findings in assays that did not include exogenous GTP, but instead relied upon guanine nucleotides retained in the membrane preparation. However, when excess GTP was included, isoproterenol-stimulated adenylate cyclase activity in MNL membranes was not altered by posture change. We conclude that substantial receptor redistribution of beta-receptors on MNL does not readily occur in physiological situations.

Summary In order to investigate properties of alpha-adrenergic receptors of human platelets, we have examined the binding of [ 3 H]yohimbine, a potent α 2 -adrenergic 3 antagonist, to intact platelets and platelet membranes. There were 207±41 [ 3 H]yohimbine sites per platelet. These had an equilibrium dissociation constant (K D ) of 2.7 ± 0.7nM, a Hill coefficient of 1.02±0.11, and were competed for by adrenergic compounds stereoselectively and with a rank order expected at α 2 -adrenergic receptors. Addition of Na + and GTP synergistically decreased the affinity of epinephrine in competing for [ 3 H]yohimbine sites in platelet membranes and the resulting affinity of epinephrine in membranes was similar to that obtained with intact platelets. Substitution of sucrose for NaCl increased the affinity of epinephrine for intact platelets. [ 3 H]yohimbine thus appears to be a useful ligand for characterization of platelet α 2 -adrenergic receptors. Our results suggest that extracellular Na + and intracellular GTP and Mg ++ may be physiological determinants of epinephrine binding to these receptors.

Important differences in binding characteristics between agonists and antagonists of the beta-adrenergic receptor have been described. However, these observations have been complicated since most available antagonists are much more lipophilic than agonists. In order to separate out those binding characteristics of agonist vs. antagonist from those characteristics of lipophilic vs. hydrophilic ligands, we have studied competition of the hydrophilic ligands isoproterenol (agonist) and CGP-12177 (antagonist) with [125I]iodopindolol binding in intact human lymphocytes. Analyzing competition curves from assays performed at 13 degrees C, 25 degrees C and 37 degrees C we demonstrated that at lower temperatures there was a decrease in IC50 for isoproterenol but not for CGP-12177. Using cells preincubated with isoproterenol then extensively washed, competition curves with both isoproterenol and CGP-12177 were biphasic, and characterized by the appearance of a population of receptors with a low affinity for both hydrophilic ligands. Furthermore, at lower temperatures the biphasic nature of these curves was accentuated and was characterized by a 6-fold and 40-fold increase in the apparent KD of a population of low affinity sites for isoproterenol and CGP-12177, respectively.

Radioligand binding to adrenergic receptors has become an important technique for identification and quantitation of these receptors. In this article we review our recent findings using subtype-selective radioligands for studying alpha 1 (e.g., [3H]prazosin) and alpha 2 (e.g. [3H]yohimbine) receptors in human platelets and rat renal cortical membranes, and we report new results on changes in adrenergic receptors of patients and animals with pheochromocytoma. Five patients with pheochromocytoma were tested prior to surgery. The number of alpha 2-adrenergic receptors on platelets from these patients and the affinity of these receptors for [3H]yohimbine were similar to values for control subjects. In rats with a transplantable pheochromocytoma we found a 50-fold increase in plasma norepinephrine levels. The number of renal cortical beta-adrenergic receptors (identified using [125I]iodohydroxybenzylpindolol) and alpha 1-adrenergic receptors were decreased while the number of alpha 2-adrenergic receptors did not change. Receptor affinities for radioligands were unaltered in animals with pheochromocytoma. These findings indicate that pheochromocytoma is associated with selective decreases in the number of renal beta- and alpha 1-adrenergic receptors without changing alpha 2-adrenergic receptor number or affinity. Furthermore, the number and affinity of alpha 2-adrenergic receptors are not altered in platelets of patients with pheochromocytoma. We conclude that down regulation (an agonist-mediated decrease in receptor number) occurs in vivo for some, but not all, types of adrenergic receptors.

Studies in clinical and experimental hypertension have identified alterations both in intracellular [Na+] and in response to hormones and neurotransmitters. We propose a hypothesis that links these two alterations. Based on recent data showing that changes in intracellular [Na+] can alter the affinity and function of platelet alpha2-adrenergic receptors, we hypothesize that elevated intracellular [Na+] in hypertension leads to enhanced response at membrane receptors. This enhancement in response to hormones and/or neurotransmitters could then contribute to the development and maintenance of the hypertensive state. Because a variety of membrane receptors are Na+-sensitive (e.g., adrenergic, muscarinic cholinergic, opiate, angiotensin, dopamine, histamine H1), this mechanism may be operative at one or more receptor types located in tissues critical to the pathophysiology of hypertension.

To determine the specificity of the previously demonstrated competition of verapamil with radioligand binding to alpha-adrenergic receptors, we examined the interaction of calcium channel blockers with alpha 1- and alpha 2-adrenergic receptors on several tissues. Verapamil competed for [3H] prazosin binding to alpha 1-adrenergic receptors and for [3H]yohimbine binding to alpha 2-adrenergic receptors in several tissues (human platelets, rat kidney and heart, and cultured muscle cells) with dissociation constants of 0.6-6 microM. The calcium channel blockers D600, D591, fendiline, and prenylamine--which are structural analogues of verapamil--also competed for [3H]yohimbine binding to human platelets. Two other calcium channel blockers, diltiazem and nifedipine, did not compete for [3H] yohimbine binding to human platelets or [3H]prazosin binding to membranes prepared from rat ventricles. We used [3H]nitrendipine binding to identify putative calcium channels on rat myocardial membranes. Nifedipine and verapamil blocked these [3H]nitrendipine-binding sites on ventricular membranes, but epinephrine and prazosin did not, indicating that the ventricular alpha 1 receptors and calcium channels are distinct. We found no specific [3H]nitrendipine binding to human platelets. We conclude that the interaction of verapamil with alpha-adrenergic receptors is not receptor subtype or tissue specific, that interaction with alpha-adrenergic receptors is not a property of all calcium channel blockers, and that the interaction of verapamil with alpha-adrenergic receptors and its interaction with calcium channels occur at at least two distinct sites.

THE catecholamines norepinephrine and epinephrine are key regulators of many physiologic events in human beings; norepinephrine acts primarily as a neurotransmitter released from sympathetic-nerve ...

We adapted a technique for isolation of mononuclear leukocyte (MNL) subsets with immunomagnetic beads to the study of beta-adrenergic receptors. Mixed MNL cells were sequentially incubated with monoclonal antibodies specific for certain MNL subsets. Sheep antimouse antibodies coupled to magnetic beads were then added, and the desired MNL subset was pulled out with a magnet. This method yielded subsets with high purity and did not alter beta-receptor density or function. Healthy volunteers were treated for 7 days with the beta 2-selective agonist terbutaline (5 mg t.i.d.). Terbutaline treatment decreased beta-receptor number and isoproterenol-stimulated cyclic adenosine monophosphate (cAMP) generation in natural killer cells, helper T cells, and suppressor/cytotoxic T cells but not in B cells. The decrease was greatest in suppressor/cytotoxic T cells and least in helper T cells. Thus beta-adrenergic receptor regulation by agonists appears to differ among MNL subsets.

We studied platelets of patients with the genetic disorder pseudohypoparathyroidism (PHP) to test whether the nucleotide-binding proteins mediating stimulation of adenylate cyclase (termed N s ) are identical to those mediating inhibition of cyclase (termed N i ). Functional responses to hormones that work through stimulation of adenylate cyclase are blunted in PHP patients. The erythrocytes of many of these patients (PHP-Ia) have previously been shown to have decreased N s activity whereas those of other PHP patients (PHP-Ib) have normal N s activity. We find that this decreased N s activity (measured by the ability to restore adenylate cyclase activity to membranes prepared from S49 cyc - cells) also occurs in the platelets of PHP-Ia but not of PHP-Ib patients. Platelets from both groups of patients accumulate less cAMP in response to prostacyclin than do platelets from control subjects. In contrast to the decreased N s function in patients with PHP-Ia, we find that N i function in platelets is similar in these patients and control subjects in several types of experiments: ( i ) epinephrine-mediated inhibition of prostacyclin-stimulated cAMP production in intact platelets; ( ii ) the affinity of platelet α 2 -adrenergic receptors for epinephrine, as determined by competition for [ 3 H]yohimbine binding; ( iii ) the decrease in receptor affinity for epinephrine produced by Na + and GTP; and ( iv ) the concentration dependence of GTP for decreasing the affinity of these receptors for epinephrine. Because N i is expressed normally in platelets from patients that are genetically deficient in N s , we conclude that N s and N i are likely to be distinct gene products.