Characterization of α2-adrenergic receptors in human platelets by binding of a radioactive ligand [3H]yohimbine

148

Biochimica etBiophysica Acta, 676 (1981) 148-154

Elsevier/North-HollandBiomedicalPress BBA 29679 CHARACTERIZATION OF a2-ADRENERGIC RECEPTORS IN HUMAN PLATELETS BY BINDING OF A RADIOACTIVE LIGAND [ 3H]YOHIMBINE AMALMUKHERJEE Department of Internal Medicine (Cardiology DivisionJ, University of Texas Health Science Center, Dallas, TX 75235 (U.S.A.J

(Received December 18th, 1980)

Key words: a2-Adrenergic receptor," Yohimbine; (Human plateletJ

[ 3H ]Yohimbine, a potent a2 oadrenergic antagonist, was used to label the ot-adrenergic receptors in membranes isolated from human platelets. Binding of [3H]yohimbine to platelet membranes appears to have all the characteristics of binding to c~-adrenergic receptors. Binding reached a steady state in 2 - 3 rain at 37°C and was completely reversible upon the addition of excess phentolamine or yohimbine (both at 10 -s M; tl/2 = 2.37 rain). [3H]Yohimbine bound to a single class of noncooperative sites with a dissociation constant of 1.74 nM. At saturation, the total number of binding sites was calculated to be 191 fmol/mg protein. [3H]Yohimbine binding was stereospecifically inhibited by epinephrine: the ( - ) isomer was 11-times more potent than the (+) isomer. Catecholamine agonists competed for the occupancy of the [aH]yohimbine-binding sites with an order of potency: clonidine > (-)-epinephrine > (-)-norepinephrine > > (-)-isoproterenol. The potent a-adrenergic antagonist, phentolamine, competed for the sites whereas the/3-antagonist, (_+)-propranolol, was a very weak inhibitor. 0.1 mM GTP reduced the binding affinity of the agonists, while producing no change in antagonist-binding affinity. Dopamine and serotonine competed only at very high concentrations. Similarly, muscarinic cholinergic ligands were also poor inhibitors of [3Hlyohimbine binding. These results suggest that [3H]yohimbine binding to human platelet membranes is specific, rapid, saturable, reversible and, therefore, can be successfully used to label a2-adrenergic receptors. Introduction a-Adrenergic receptor binding sites have been demonstrated in human platelet membranes using antagonists such as [3H]dihydroergocryptine and [3H]phentolamine [1-4]. There is considerable experimental evidence demonstrating the existence of two types of a~adrenergic receptors. These are classified as postjunctional (at-adrenergic) and prejunctional or neuronal (a2-adrenergic) [5-7]. However, Berthelson and Pettinger [8] have shown that the az-adrenergic(neuronal) receptors also exist in many nonneuronal tissues. A wide variety of drugs have been identified by physiological experiments and binding studies to discrimintate between the receptor sub-types by their relative binding affinities [9-11 ]. Analyzing the binding data in several tissues by corn-

puter-modeling techniques, Hoffman et al. [10] have proposed that dihydroergocryptine and phentolamine have approximately equipotent affinity for a~- and a2-adrenergic receptors whereas prazosin and yohimbine are potentially cq- and a2-adrenergic-selective drugs, respectively. Previous studies [ 12-14] have shown that yohimbine is highly potent in blocking epinephrine-induced pletelet aggregation, whereas phenoxybenzamine (an aFadrenergic-selective drug) is ineffective, Newman et al. [2] and Hofman et al. [10] have also shown that [3H]dihydroergocryptine binding to human and rabbit platelets was potently inhibited by selective a2-adrenergic drugs. This suggested that platelets might be considered to have predominantly a2-type adrenergic receptors. The purpose of the present study was to charac-

0304-4165/81/0000-0000[$02.50 © Elsevier/North-HollandBiomedicalPress

149 terize the a2-adrenergic receptor binding sites in platelets using the selective a2-adrenergic drug, [aH]yohimbine. The data obtained demonstrated that the binding of [aH]yohimbine to human platelet lysates has all the characteristics, (i.e., specificity, stereospecificity, saturability and reversibility) which would be expected of binding to physiological a-adrenergic receptors. Materials and Methods

Matetqals. [3H]Yohimbine (86.2Ci/mmol) and [aH]dihydroergocryptine (38.8 Ci/mmol) were obtained from New England Nuclear, Boston, MA. Other compounds used in this study were from the following sources: Sigma Chemical Co., St. Louis, MO: (-)-epinephrine bitartrate, (-)-norepinephrine bitartrate, (-)-isoproterenol bitartrate, GTP, 5'-guanylimidodiphosphate and yohimbine hydrochloride; Wintthrop Sterling Drug, NY: (+)-epinphrine bitartrate and (+)-norepinephrine bitartrate; Boehringer Ingelheim, NY: clonidine hydrochloride; Pfizer, Groton, CN: prazosin. Platelet membrane preparation. 20-30ml from each normal healthy volunteer (males and females, age 25-35 years) were mixed with 1 ml acid citrate dextrose. Platelet-rich plasma was prepared by centrifugation at 150 Xg for 10 min at 25°C in a Beckman TJ6 centrifuge. Platelet-rich plasma was centrifuged at 1 500 Xg for 10 min at 25°C. Platelets were washed twice according to the procedure of Alexander et al. [3] in a buffer containing 138 mM NaC1, 5 mM KC1, 8 mM Na2HPO4, 2 mM NaH2PO4, 10 mM EDTA-Tris (pH 7.2). Washed platelets were subjected to two cycles of freeze-thawing in liquid nitrogen and followed by glass-Tefion homogenization. The particulate fraction was then centrifuged at 40000Xg for 15 min at 4°C in a Beckman J21 centrifuge. The supernatant was discarded and the particulate fraction, after being washed twice in cold Tris-saline buffer (138 mM NaC1, 5 mM MgCI2, 1 mM EGTA-Tris and 25 mM Tris-HC1, pH 7.4) by resuspension and centrifugation, was suspended in the same buffer at a protein concentration of 0.3-0.5 mg/ml. ~-Adrenergic receptor assay in platelet membranes. Membrane protein (200-400/.tg/ml) was incubated with six to seven different concentrations of [aH]yohimbine (0.2-10 nM)in a total volume of 150/,tl

of Tris-saline buffer at 37~C for 10 min. The incubation was terminated by diluting the incubation mixture rapidly with 2 ml ice-cold Tris-saline buffer, followed by rapid vaccum filtration through Whatman GFC filters. The filters were washed rapidly by a vacuum filtration with four 5-ml portions of ice-cold Tris-saline buffer. After drying, the filters were placed directly into a Triton/toluene-based scintillation cocktail and counted in a Backman LS7500 counter, at 45% efficiency. In each experimenta, nonspecific binding to platelet membranes was determined by measuring the amounts of radioactivity retained on filters when incubations were performed in the presence of either 10 -s M yohimbine or 10 -s M phentolamine. Specific binding was determined by subtracting nonspecific from total binding and was usually between 60-80%. Using these direct binding methods, the number and affinity of yohimbine-binding sites in the platelet membrane were analyzed according to the method of Scatchard [15], fitting the regression lines by the method of least squares. Calculation of apparent dissociation constant for various compounds. The apparent dissociation constant (Ka) of competing agents for binding sites was calculated from the following equation of Cheng and Prusoff [16]: ECso Ka = 1 +S/Ka (3H]yohimbine) where Ka is the apparent dissociation constant of competing agents, Kd(13Hlyohirnbine) is the equilibrium dissociation constant of [aH]yohimbine determined from kinetic studies (1.17 nM), S is the concentration of [3H]yohimbine present in the binding assay (6-7 nM) and ECso is the concentration of competing agents causing 50% inhibition of specific [aH]yohimbine binding. Protein was assayed by the method of Lowry et al. [17] using crystalline bovine serum albumin as standard.

Results

Kinetic of binding. Binding of [aH]yohimbine to human platelet membranes was rapid, reaching equilibrium within 2-3 rain at 37°C (Fig. 1). The pseudo-

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Fig. 1. The time course of [3H]yohimbine binding to human platelet membranes. [3H]Yohimbine, with and without 10 -s M unlabeled yohimbine, was preincubated in buffer for 1 rain and at the specified intervals after the addition of membranes, 0.1 ml samples were removed, diluted in 2 ml ice-cold assay buffer and filtered. Specific binding is defined as the difference between incubation mixtures with 10 -5 M unlabeled yohimbine and otherwise identical mixtures without unlabeled yohimbine. The results are the means of duplicate determinations from three separate experiments. (Inset) Pseudo-first-order kinetic plot of the [3H]yohimbine-binding data presented in the main figure. X represents the amount of [3H]yohimbine bound at each time (t) and Xeq is the amount bound at steady state, kob is the reversible pseudofirst-order rate constant and is equal to the slope of the line.

first-order rate constant, kob , for the association reaction was obtained from the slope in Fig. l(inset) and was found to be 1.46 min -1. Fig. 2 shows the reversibility of binding. The rate of dissociation of specifically bound [3H]yohimbine from platelet membranes was determined by adding excess unlabeled yohimbine (10 -s M) to the equilibrated mixture of [3H]yohimbine and platelet membranes. Dissociation was rapid (Tw2 = 2.37 min) and first order characterized by the rate constant kz of 0.242 min-l(Fig. 2,inset). The second-order assication rate constant, kl, was then calculated from the equation: kl = ( k o b - kz)/ [yohimbine], where [yohimbine] is equal to the concentration of yohimbine in the reaction (5.9 nM). k, for the reaction of [3H]yohimbine with platelet membrane was 0.207 nM-~' min-t. The ratio of the rate constants, k2/k,, provides an estimated of the equilibrium dissociation constant, Kd, for the interaction between [3H]yohimbine and the platelet membranes. From the values of 0.207 nM-1' min -~ for kl and 0.242 rain -1 for k2, the determined Kd was 1.17 nM. "1o

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Fig, 2. The time course of [3H]yohimbine binding to human platelet membranes. Membranes were incubated with [3H]yohimbine for 15 rain which represents zero time in this figure. At zero time, 10 -5 M unlabeled yohimbine was added to the incubation mixture and specific binding was determined at various time intervals. Maximal binding refers to the specific binding just prior to the addition of unlabeled yohimbine at zero time. The values are the means of duplicate determinations from three separate experiments. (Inset) First-order rate plot of dissociaion of [3H]yohimbine binding. The slope, determined by linear regression analysis, is equal to the first-order reverse (k 2) rate constant.

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Fig. 3. Specific binding of [3H]yohimbine to human platelet membranes as a function of increasing concentration of [3H]yohimbine. Membranes were incubated with various concentrations of [3H]yohimbine in the presence or absence of unlabeled yohimbine and the specific binding was determined. The values are the means of duplicate determinations from five separate experiments. (Inset) Scatchard analysis of [3H]yohimbine binding to human platelet membranes. B/F is the ratio of bound [3H]yohimbine to free [3H]yohimbine. The slope, determined by linear regression analysis, is equal to - 1 / K d. The number of binding sites is calculated from the intercept of the plot with the abscissa.

151

Saturability of binding. [aH]Yohimbine-binding sites in the human platelet membranes appeared to be saturable at concentrations of 8 - 1 0 nM of radioactive ligand as evidenced by the hyperbolic shape of the concentration-binding curve (Fig. 3). The calculated total number of binding sites from Scatchard analysis of the data [15] was 191 +-23 fmol/mg protein (mean of four experiments). The dissociation constants of the binding site was 1.74 +- 0.23 nM (Fig. 3, inset).

Inhibition of [3H]yohimbine binding by competing ligands. The binding of [3H]yohimbine was stereospecifically inhibited by epinephrine (Fig. 4 ) a n d the ( - ) isomer was found to be 1 l-times more potent thant the (+) isomer. Adrenergic agonists competed for the [aH]yohimbine-binding sites with an order of potency: clonidine > (-)-epinephrine > (-)-norepinephrine > > (-)-isoproterenol (Fig. 5). The a-adrenergic agonists had high affinity for the binding sites, with Kd values of 0.095, 0.352 and 0.704/.tM for clonidine, (-)-epinephrine and (-)-norepinephrine, respectively (Fig. 5 and Table I). In contrast, the /3-adrenergic agonist, (-)-isoproterenol, is a very weak inhibitor of [3H]yohimbine binding (Kd = 56/.tM, Fig. 5 and Table I). tx-Adrenergic antagonists competed strongly for the [3H]yohimbine-binding sites. Phentolamine, a potent a-adrenergic antagonist, competed with a Kd of 0.041 /IM (Fig. 6 and Table I). Yohimbine

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Fig. 4. Sterospecificity of the inhibition of [3H]yohimbine binding by epinephrine. Platelet membranes incubated with 5-6 nM [3H]yohimbine and varying concentrations of (-)epinephrine (. *) or (+)-epinephrine (o "' 6) for 15 min at 37°C. After incubation, samples were filtered and counted. 100% specific binding refers to binding in the presence of 10-s M unlabeled yohimbine. The values are the means of duplicate determinations from five to eight separate experiments.

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Fig. 5. Inhibition of [3H]yohimbine binding by adrenergic agonists. Platelet membranes were incubated with 5-6 nM [aH]yohimbine and varying concentrations of clonicine (~ D), (-)-epinephrine (.--------o), (-)-norepinephrine (o--------o) and (-)-isoproterenol (zx a) for 15 min at 37°C. After incubation samples were filtered and counted. 100% specific binding refers to binding in the presence of 10 -s M unlabeled yohimbine. The values are the means of duplicate determinations from two to eight separate experiments.

TABLE I THE APPARENT DISSOCIATION CONSTANTS (Kd) OF ADRENERGIC AND OTHER DRUGS DETERMINED BY COMPETITION FOR [3H]YOHIMBINE BINDING n.i., no binding with 10-2 M drug. Drug

Competition for [3H]yohimbine binding (Kd, ~M)

Clonidine (-)-Epinephrine (+)-Epinephrine (-)-Norepinephrine (-)-lsoproterenol Yohimbine Phentolamine Prazosin (+)-Propranolol Dopamine a Haloperidol a Serotonin a Oxotremorine a Atropine a

0.095 0.352 4.32 0.704 56 0.0034 0.041 6.85 10.0 4.0 3.6 15.0 n.J. n.i.

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Fig. 6. Inhibition of [JH]yohimbine binding by adernergic antagonists. Platelet membranes were incubated with 5-6 nM [3H]yohimbine and varying concentrations of unlabeled yohimbine (o ,), phentolamine (D - tJ), prazosin (~ n) and (_+)-propranol (, ,) for 15 min at 37°C. After incubation, samples were filtered and counted. 100% specific binding refers to binding in the presence of 10-s M unlabeled yohimbine. The values are the means of duplicate determinations from two to ten separate experiments.

itself competed for the binding sites with a K d of 0.0034/aM. This Kd is in reasonable agreement with the determined Kd of 0.0012/aM and Kd of 0.0017 /aM determined by equilibrium binding studies. However, prazosin, a highly specific al-adrenergic receptor antagonist [10], was a weak inhibitor of [3H]yohimbine binding (Kd = 6.85/aM, Fig. 6 and Table I). Unlike a-adrenergic antagonists, (+-) propranolol was a very weak inhibitor of binding of [3H]yohimbine (Kd = 15/aM, Fig. 6 and Table I). Dopamine had a Kd of 4.0/aM, consistent with its known a-adrenergic activity. Serotonin had a low affinity for the binding sites with a Kd of 15.0/aM. The dopaminergic antagonist, haloperidol, inhibited binding with a Kd of 3.6/aM, which is considerably higher than that of phenolamine (Kd = 0.041 /aM) and yohimbine (K d = 0.0034/aM). This is consistent with the known weak a-adrenergic antagonist activity of haloperidol [ 181 . Oxotremorine and atropine (muscarinic cholinergic receptor antagonists) did not inhibit [3H]yohimbine binding.

Effects of guanyl nulcoetide on the binding of a-adrenergic agents to the receptors. The effect of GTP on various c~-adrenergic agents was studied by their ability to displace [3H]yohimbine binding in the presence and absecne of 0.1 mM GTP. As shown in

Phentolamine Clonidine (-)-Epinephrine

Table II, GTP decreased the ability of (-)-epinphrine and clonidine to inhibit [3H]yohimbine binding. The displacement curve for (-)-epinphrine and clonidine shifted 10- and 4-fold to the right, respectively. The displacement curve for phentolamine, however, was not altered. Maximal and nonspecific binding of [3H]yohimbine were not altered by the addition of 0.1 mM GTP. Discussion

Yohimbine has been known to be a potent a2-adrenergic antagonist by virtue of its potency in blocking presynaptic a-adrenergic receptors [9]. Recently, Hoffman et al. [10] have successfully used yohimbine to displace [3H]dihydroergocryptine from c~-adrenergic receptor sites. Analyzing the displacement curve by computer-modeling techniques, Hoffman et al. [10] and Wood et al. [11] have clearly shown that the human platelets have almost exclusively c~-type adrenergic receptors. An alternative approach to study the subclass of receptors, e.g., astype adrenergic recptors, would be to label the receptors directly with a high specific activity a2-adrenergic-selective drug and then study its binding characteristics. Yohimbine, a highly selective a2-adrenergic drug, has been recently labeled with tritium; it has a very high specific activity of 86 Ci/mmol. In this communication, I report studies of [3H]yohimbine binding to human platelet membranes. [3H]Yohimbine was found to bind rapidly and reversibly. Specific binding of [3H]yohimbine to the recep-

153 tors was high (60-80%) and saturable with high affinity. The binding of [3H]yohimbine was stereoselective, (-)-epinephrine was l 1-times more potent than (+)-epinphrine. The binding of [3H]yohimbine to platelet membranes was specifically inhibited by agents in the potency order of: unlabeled yohimbine > phentolamine > chonidine > (-)-epinphrine > (-)-norepinephrine>>(-)-isoproterenol and very weakly inhibited by prazosin. These observations indicated that [3H]yohimbine bound to t~-adrenergic receptors and specifically to a2-type adrenergic receptors. The dissociation constants of yohimbine obtained by kinetic analysis of the rate of association and dissociation, by equilibrium studies and by displacement of [3H]yohimbine by various concentrations of unlabeled yohimbine were 1.17, 1.74 and 3.4nM, respectively. These results are in reasonable agreement with the data of Newman et al. [2] and Hoffman et al. [10] who found K d values of 2 and 0.8 nM, respectively, for yohimbine by their [3H]dihydroergocryptine-displacement experiments. Yohimbine has also been found to antagonize strongly (Kd --0.06 #M) the epinephrine-induced prostaglandin E~stimulated platelet membrane adenylate cyclase [2]. Scatchard analysis of [3H]dihydroergocryptine binding to the human platelet membranes gave an apparent affinity of 6.5 +- 1.1 nM (data not shown); this and the data obtained by others [1-3, 10] show that yohimbine binds to platelet membranes with much higher affinity than dihydroergocryptine, which is a nonselective ligand [ 10,11 ]. Similarly, phentolamine, also a nonselective ligand [10,11 ], bound to platelet membranes with much lower affinity (7-14 nM) than yohimbine [1,2,4]. The number of binding sites in human platelet membranes obtained by [3H]yohimbine binding (191 fmol/mg protein) is quite comparable to the data obtained on [3 H] dihydroergocryptine binding (130-180 fmol/mg protein) by others [2,3]. Using [3H]phentolamine to label ct-adrenergic receptors in human platelets, Steer et al. [4] have found the receptor number to be 165 fmol/mg protein. This value is also in reasonable agreement with our value obtained by [3H]yohimbine binding: Considering that phentolamine and dihydroergocryptine label al- and a2-adrenergic receptors with equal affinity [10,11], the maximal number of binding sites (130-180 fmol/mg protein) with these agents

represents total adrenergic receptors in human platelet membranes. Since yohimbine has been characterized as an ct2-adrenergic-selective drug and possesses a comparable number of binding sites (191 fmol/mg protein) to [3H]phentolamine and [3H]dihydroergocryptine found with [3H]yohimbine, it is reasonable to suggest that a-adrenergic receptors in human platelet membranes are almost exclusively the a2-adrenergic type. Similar findings have also been reported by Hoffman et al. [10] by computer analysis of the data of inhibition of [3H]dihydroergocryptine binding to human platelet membranes by yohimbine. The binding of [3H]yohimbine was inhibited by a number of a2-adrenergic agonists and antagonists. Clonidine was found to be the most potent agonist with a Ka of 0.041 /~M. Yohimbine was the most potent antagonist (Ka = 0.0034/.tM). The dissociation constants and the order of potency of the adrenergic ligands tested by measuring the inhibition of [3H]yohimbine binding to the platelet membrane are in reasonable agreement with the data that have been obtained by studying either [3H]dihydroergocryptine or [3H]phentolamine inhibition by the same drugs [2-4]. These observations suggest that all three ligands, [3H]yohimbine, [3H]dihydroergocryptine and [3H]phentolamine, may bind to the same site(s) in the platelet membrane. The same sites are also apparently capable of binding many t~-adrenergic agonists and antagonists. The effects of GTP on the binding of a-adrenerglc agents to the receptors were also studied. The observations indicate that GTP markedly decreased the ability of (-)-epinephrine and clonidine to inhibit [3H]yohimbine binding while no inhibition by phentolamine was noted. These results are in complete agreement with the data of Tsai and Lefkowitz [19] and Steer et al. [4] who observed inhibition of [3H]dihydroergocryptine of [aH]phentolamine binding in human platelests by agonists in the presence of GTP. It has also been shown by Jacobs et al. [20] that the presence of GTP is absolutely necessary for inhibition of platelet adenylate cyclase by a-adrenergic agonists. Whether this paradoxical effect of GTP is mediated by two different nucleotide binding sites, inhibitory and stimulatory, as suggested by Yamamura et al. [21], remains uncertain. Thus, high specific activity [3H]yohimbine has

154 been successfully used in the present experiments to label human platelet membrane a2-adrenergic receptors with a much higher affinity than previously used ligand, such as [3H]dihydroergocryptine and [3H]phentolamine. Also, the fast kinetics of association and dissociation o f [3H]yohimbine make it an ideal ligand to use in receptor binding studies, since the physiological hormone-receptor interaction is known to be a rapid process. A recent paper [22] has reported results similar to those o f this study.

Acknowledgments This work was supported by the National Institute of Health Ischemic Heart Disease Specialized Center o f Research Grant, HL-17669, the Harry S. Moss Heart Fund, and a grant from the American Heart Association, Texas Affiliate. The Author Acknowledges Dr. James T. Willerson for constant encouragement, support and for reading the manuscript. The technical assistance o f Mr. Arun Bellary, Mr. Matthew Hogan and Mrs. Joan Cary, and the secretarial assistance o f Ms. Laurie Grey are also appreciated.

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4 Steer, M.L., Khorana, J. and Galgoci, B. (1979) Mol. Pharmacol. 16,719-728 5 Starke, K., Taube, H.D. and Borowski, G. (1977) Biochem. Pharmacol. 26,259-268 6 Starke, D., Endo, T. and Taube, H.D. (1975) Nature 254, 440-441 7 Langer, S.Z. (1974) Biochem. Pharrmacol. 23, 17931800 8 Berthelson, S. and Pettinger, W.A. (1977) Life Sci. 21, 595 -606 9 Starke, K. (1977) Rev. Physiol. Biochem. Pharmacol. 77, 1-124 10 Hoffman, B.B., DeLean, A., Wood, C.L., Schocken, D.D. and Lefkowitz, R.J. (1979) Life Sci. 24, 1739-1746 11 Wood, C.L., Arnett, C.D., Clark, W.R., Tsai, B.S. and Lefkowitz, R.J. (1979) Biochem. Pharmacol. 28, 12771282 12 Barthel, W. and Markwardt, F. (1974) Biochem. Pharmacol. 24, 37-46 13 O'Brien, J.R. (1963) Nature 763-765 14 Mills, D.C.B. and Roberts, G.C.K. (1967) J. Physiol. 193, 443-453 15 Scatchard, G. (1949) Ann, N.Y. Acad. Sci. 51,660-672 16 Cheng, Y. and Prusoff, W. (1973) Biochem. Pharmacol. 22, 3099-3108 17 Lowry, O.H., Rosebrough, N.J., Farr, ,~L. and Randall, R.J. (1951) J. Biol. Chem. 193,265-275 18 U'Prichard, D.C., Greenberg, D.A. and Snyder, S.H. (1977) Mol. Pharmacol. 13,454-473 19 Tsai, B.S. and Lefkowitz, R.J. (1979) Mol Pharamacol. 16, 61-68 20 Jacobs, K.H., Saur, W. and Schultz, G. (1978) FEBS Lett. 85,167-170 21 Yamamura, H., Lad, P.M. and Rodbell, M. (1977) J. Biol. Chem. 252, 7964-7966 22 Motulsky, H.J., Shartil, S.J. and Insel, P.A. (1980) Biochem. Biophys. Res. Commun. 97, 1562-1570