Br. J. Pharmacol. (1988), 93, 817-826

Characterization of rat liver f-adrenoceptors during perinatal development as determined by [1251]-iodopindolol radioligand binding assays

1 Keith Snell & Carole A. Evans

Department of Biochemistry, Metabolic Regulation and Receptor Research Group, University of Surrey, Guildford, Surrey, GU2 5XH

1 The subtype specificity of P-adrenoceptors in foetal (20 days post coitum) rat liver membrane preparations has been determined by use of [1251]-iodopindolol binding assays and the character- istics of radioligand binding have been resolved. 2 The kinetics of radioligand association and dissociation (in the presence of 5 x 1O-4M isoprenaline) showed an association rate constant of 1.5 x 10 M- s1- and dissociation rate con- stant of 9.1 x 10-4 s 1, corresponding to a dissociation constant for [I251]-iodopindolol of 60.7 pM. A similar dissociation constant (75 pM) was determined by saturation binding assays. 3 The rank order of potency for displacement of [1251]-iodopindolol binding was consistent with binding to a predominantlyf2-adrenoceptor population (i.e. ICI 118551 > isoprenaline > adrena- line > noradrenaline > ). Computer analysis of displacement curves in the presence of a f,-subtype selective agent (atenolol) or a 2-subtype selective agent (ICI 118551) revealed the pre- sence of P2- and fl1-adrenoceptor subtypes in a ratio of about 80: 20%. 4 Saturation binding assays by use of ['25I]-iodopindolol were carried out at different perinatal ages to determine total f-adrenoceptor concentrations and fi2-subtype (in the presence of 5 x 1i-' M atenolol) adrenoceptor concentrations. Competition binding assays with atenolol confirmed that at all ages apparent #2-adrenoceptor binding accounted for 84-95% of the total P-adrenoceptor binding. The total fl- and fJ2-adrenoceptor binding capacity increased by 2.3 fold from 20 days post coitum to birth, and then decreased postnatally at 1 and 2 days post partum. The dissociation constant for ['25I]-iodopindolol binding did not show any change with age. 5 The change in fi2-adrenoceptor concentration with age is discussed in relation to the changing ,B-adrenoceptor-mediated responsiveness of glucose production by rat liver during perinatal devel- opment.

Introduction Catecholamines play a critical role in a number of 1982). At this time there is a refractoriness in the essential developmental adaptations that occur at liver to the stimulating effects of glucagon on glyco- the time of birth. For example, foetal lung matu- genolysis (Blazquez et al., 1976; Snell & Walker, ration (Davis et al., 1987) and perinatal hepatic 1978), which emphasizes the important role of cate- glucose production (Sperling et al., 1984) are pro- cholamines in this metabolic process at birth. cesses under the control of endogenous catechol- The physiological responses of target tissues to amine concentrations in the blood. In the rat there catecholamines are mediated by binding to a- and are high concentrations of plasma adrenaline and fi-adrenoceptors. In foetal and neonatal rat liver, in noradrenaline at birth, which rapidly decrease to low sharp contrast to adult male rat liver, metabolic levels before showing significant secondary increases responses to adrenoceptor agonists appear to be at a time (2h post partum) which coincides tempo- mediated by fi-adrenoceptors (Sherline et al., 1974; rally with the mobilization of liver glycogen for Moncany & Plas, 1980; Huhn et al., 1983). However, increased hepatic glucose production (Cuezva et al., caution is needed in the interpretation of some of these findings which have used hepatocytes in 'Author for correspondence. culture, in view of the recent demonstrations of a © The Macmillan Press Ltd 1988 818 K. SNELL & C.A. EVANS rapid shift from a- to #-adrenoceptors when adult NaCl, 20 mm Tris-HCl buffer (pH 7.5), 2mM MgCl2, rat hepatocytes are cultured in vitro (Nakamura et 1 mm ascorbic acid, and various concentrations of al., 1983; Kunos et al., 1984). The characterization of [125I]-iodopindolol and of competing drugs where hepatic adrenoceptors by radioligand binding studies appropriate. The reaction was started by addition of in the perinatal rat has received little attention, and liver membrane preparation (0.20-0.40 mg of protein) the single study described in the literature and was continued for 45 min for saturation binding (McMillian et al., 1983) was limited in the extent of assays, for varying times up to 45 min when studying measurements made during the perinatal period. The association kinetics, or for 45 min followed by aim of the present study was to characterize the varying times up to 20 min after addition of dis- nature of the f$-adrenoceptor subtype in perinatal rat placing drug (5 JM, final concentration) when study- liver and to investigate possible changes in fi- ing dissociation kinetics. The reaction was adrenoceptor number and ligand affinity during late terminated by the addition of 2 ml of ice-cold wash foetal and early neonatal life by use of [125I]- buffer (145 mm NaCl, 10mM Tris-HCI, pH 7.5), fol- iodopindolol as a radioligand for the binding studies. lowed by immediate and rapid filtration through Whatman GF/C glass fibre filters (Whatman Lab Sales Ltd., Maidstone, Kent) using a Millipore 1225 vacuum filtration manifold (Millipore (UK) Ltd., Methods Harrow, Middlesex). The incubation tubes were rinsed with 2 ml of ice-cold wash buffer and the filters were washed with this and with 2 x 5 ml of Cell membrane preparation ice-cold wash buffer. Membrane-bound [125I]_ iodopindolol trapped on the filters was counted in a AHA strain rats (200-300 g body wt) from the breed- Searle Analytic Inc. 1185R gamma counter. Specific ing unit of Glaxo Research Ltd were mated over- non-displaceable binding was defined as the differ- night and certified pregnant on recovery of vaginal ence between binding in the absence (total binding) plugs on day 1 post coitum. Foetal rats, at the gesta- and in the presence of 2 x 10-4M (-)-isoprenaline tional ages indicated in Results, were rapidly (Nahorski & Richardson, 1979). In all saturation delivered by caesarian section of unanaesthetized binding assays Scatchard analyses were based on pregnant dams killed by cervical dislocation. Partur- data derived from measurements at 10-12 concentra- ition occurred naturally between days 21 and 22 post tions of radioligand from 10-600pM. In competition coitum, and newborn rats were used immediately displacement studies Ki values were determined from after the birth of the complete litter, or 24 and 48 h IC50 values derived from Hill plots as described by later, for receptor binding studies. Foetuses and Cheng & Prusoff (1973). In these studies the radioli- neonates were decapitated, the livers were rinsed in gand was added at 70 pM, corresponding to the ice-cold homogenization medium (145 mM NaCl, determined KD for this ligand (see Results). In the 2 mm MgCl2, 20 mm Tris-HCl, pH 7.5), and then same studies computer-assisted iterative curve-fitting homogenized in about 40 vol of homogenization procedures based on models with one, two or three medium by a Silverson Vortmix homogenizer fol- non-interacting binding sites were carried out to lowed by a Dounce homogenizer. Cell debris and determine the relative proportions of fi-adrenoceptor unbroken cells were removed by centrifugation at subtypes as described by Hancock et al. (1979). 2000g for 10min, and the supernatant was centri- fuged at 40000g for 15min. This membrane prep- Materials aration was resuspended in fresh homogenizing medium by the Dounce homogenizer and recentri- [125I]-iodopindolol was prepared and purified by fuged at 40000 g for 15min. The washed membrane the method of Barovsky & Brooker (1980) by iodina- preparation was then resuspended in storage tion of (-)- and was a gift from N. Cook medium (250 mm sucrose, 5 mM MgCl2, 50mM Tris- and S.R. Nahorski, Department of Pharmacology HC1, pH 7.5). Aliquots of the preparation were used and Therapeutics, University of Leicester. The freshly prepared or were snap-frozen in liquid N2 radioligand was stored at -60°C and was used and used after storage at - 60°C. Stored frozen prep- within 6 weeks of preparation. (-)-l-[Propyl-2,3- arations produced results identical to fresh prep- 3H]-dihydroalprenolol and (-)-[1125]-iodo- arations. were obtained from Amersham International plc (Amersham, Bucks.). Atenolol, ICI Radioligand binding assays 118551 (D,L-erythro-3-isopropylamino- 1 -[7-methyl- 4-indanyloxy]-2-butanol hydrochloride), ketanserin, Incubations (150p1) in triplicate were carried out at atropine sulphate, spiperone, mepyramine, raniti- 25°C in polypropylene tubes containing 145 mm dine, and phentolamine mesylate were gifts from fi-ADRENOCEPTORS IN PERINATAL LIVER 819

Glaxo Group Research Ltd. (Ware, Herts.). (-) binding assay subsequently developed. A time course Isoprenaline bitartrate, (±)-adrenaline, (+)- of association showed a hyperbolic relationship with noradrenaline-HCI, (±)--HCI, Tris and time for total and specific binding, reaching a all other reagents were of highest purity available plateau at 25min (Figure 1). Since less than 10% of from Sigma Chemical Co. (Poole, Dorset). the total radioligand present was specifically bound at equilibrium, the association reaction shows pseudo-first order kinetics and an association rate Results constant of 1.5 x 107M-1 s1 was determined from the linear plot of log [Be/Be]-[B,] versus time (where Be is the concentration of ligand specifically Kinetics of["j25]-iodopindolol binding infoetal rat bound at equilibrium and [BJ is the concentration liver specifically bound at time t in s). The kinetics of radioligand dissociation were The characteristics of binding of ['25I]-iodopindolol studied, after equilibrium binding was attained were determined in liver membrane preparations (45 min), by the addition of 5 x IO- M isoprenaline. from foetal rats at 20 days post coitum. These charac- [1251]-iodopindolol binding dissociated in a mono- teristics have not previously been described for foetal phasic manner with a half-life of 12.7 min and with a liver and are an essential validation of the saturation dissociation rate constant of 9.1 x 10-4s- as deter-

1.6 -

x 1.2 - C 0

0. 0)O 1/ E E 0.8 -

0 .0

N 0

Incubation period (min) Figure 1 Time-course of association of [125I]-iodopindolol with a foetal rat liver membrane preparation. Foetal (20 days post coitum) rat liver membranes were prepared as indicated in Methods, and incubated for the times indicated with 70pM [125I]-iodopindolol. Specific radioligand binding (El) was calculated as the difference between total binding (0) in the absence of 2 x 10-'M (-)-isoprenaline and binding in the presence of the #-agonist and is expressed as mol mg-1 membrane protein x I0 ". Each point is the mean of triplicate determinations. Inset shows the secondary plot of log (Be/Be - B) against incubation period in s, where Be is the concentration of radioligand specifically bound at equilibrium and B, is the concentration specifically bound at time t in s. 820 K. SNELL & C.A. EVANS

3.0r

2.51-

x c ._ 0 2.0h 4-

CD E 'a E 1.5 1- 'D c :3 0 .0

Z 0 m

._ 1.01- Cl 0 0o 0 0

C)I5 - Time (min)

o _ L 0 5 10 15 20 Time after isoprenaline addition (min) Figure 2 Time-course of dissociation of ['251]-iodopindolol with a foetal (20 days post coitum) rat liver membrane preparation. Total (0) and specific (U) radioligand binding was as defined in Figure 1. Dissociation of radioligand binding was determined for different time periods after equilibrium binding of 70pM ['25I]-iodopindolol was achieved (45min), by the addition of excess (-)-isoprenaline (5 x 10-4M). Each point is the mean of triplicate determinations. Inset shows the secondary plot of log (Be/BJ against time, where Be and B. are as defined in the legend to Figure 1. mined from the linear plot of log [Be/BJ versus time (2.1 x 10- 7 M) > (±)-adrenaline (3.5 x 10-7M) > (Figure 2). From the rate constants of association (± )-noradrenaline (5.1 x 10-6 M) > atenolol (1.4 and dissociation, a dissociation constant (KD) for x 10 5M) which is consistent with the binding of [125I]-iodopindolol of 60.7 pM was determined. iodopindolol to a predominantly fi2-adrenoceptor Compounds with known affinity for f- population (see Williams & Lefkowitz, 1978). adrenoceptors displaced equilibrium-bound [1251]_ Various compounds with known pharmacological iodopindolol from foetal liver membrane activity other than for fi-adrenoceptors were also preparations in a concentration-dependent manner tested for their ability to displace radioligand and (Figure 3). The rank order of displacing potency had no significant effect when present at 10-5- (with Ki values in parentheses) for these compounds 10-6m: phentolamine (x-adrenoceptor), ketanserin was ICI 118551 (5.6 x 10-9M) > (-)-isoprenaline (5-hydroxytryptamine receptor), atropine (muscarinic fl-ADRENOCEPTORS IN PERINATAL LIVER 821

a 75 100 'a 100 CLa o 80 V 0 80O

N 60

0 60 H c 40 a1)E 401- O 20 'aso U 0 0 8 7 6 5 'a0 -log [Displacing ligand] (M) t0o I I Figure 3 Competition displacement of [125I]_ 5 6 7 8 9 10 iodopindolol binding to foetal (20 days post coitum) rat liver membrane preparations. [125I]-iodopindolol was present at a concentration of 70 pM and displacing °b E) 100r_ agents used were: ICI 118551 (0), (-)-isoprenaline E (@), (±)-adrenaline (El), (±)-noradrenaline (a), and atenolol (A). Binding is expressed as the percentage of [125I]-iodopindolol displaced by the competing agent X 80 compared to the binding in the absence of competing ._ agent. The isotherms shown are those representative of two or three separate experiments; each point is the 60 _ mean of triplicate determinations.

40 - cholinoceptor), spiperone (D2-dopamine receptor), mepyramine (H1-histamine receptor), ranitidine (H2-histamine receptor). 20 _ The appearance of the atenolol displacement curve 0 shown in Figure 3 suggests that displacement of 0 L L I I I radioligand is not from a single class of specific 5 6 7 8 9 binding site within the tissue preparation. This was -log [Displacing ligand] confirmed by analysis of the data by use of the (M) Eadie-Hofstee transformation (Figure 4). It can be Figure 4 Eadie-Hofstee transformation of competition seen that, whereas the non-subtype selective agonist of [125I]-iodopindolol binding from foetal (20 days post isoprenaline showed a linear relationship, the coitum) rat liver membrane preparations by (a) (-)- selective a isoprenaline and (b) atenolol. The displacement of /,B-subtype antagonist (atenolol) showed binding by the competing agents is expressed as a per- curvilinear relationship. This suggests that the foetal centage of binding in the absence of competing agent liver membrane preparation does not contain a and is plotted against the ratio of % displacement to single homogeneous population of receptor corre- the concentration of the competing agent added in M sponding to one subtype or the other. Analysis of the units. ['251]-iodopindolol was present at 70pM. Each Hofstee plots by the method of Rugg et al. (1978) for point is the mean of triplicate determinations in each the data with atenolol revealed that about 20% of experiment, and the plots shown are representative of the binding sites present had affinity for this three separate experiments. 3,B-selective antagonist. Although this procedure has been suggested to lead to overestimation of the pro- portion of the high-affinity component (Hancock et data from the displacement curves in the presence of al., 1979), it has been shown to provide an adequate the /,-subtype and 132-subtype selective agents, by assessment when antagonists of adequate (> 30 fold) the methods described by Hancock et al. (1979). This subtype selectivity are used (Rugg et al., 1978; Dick- analysis revealed that in both cases the experimental inson et al., 1981). Nevertheless, further character- data were best described by theoretical curves corre- ization of /-adrenoceptor subtype proportions was sponding to a two-site binding model. Assuming carried out by computer-assisted curve-fitting of the these to be the il- and P2-subtype receptors, the cal- 822 K. SNELL & C.A. EVANS

24

20

E 16 M. -om c

.0 -5 12 'a~0 Q

._ 0 Ni 8

0 0 100 200 300 400 500 600 [[12511]-iodopindololJ (pM) Figure 5 Saturation binding isotherm of increasing concentrations of ['251]-iodopindolol to foetal (20 days post coitum) rat liver membrane preparation. Specific radioligand binding (EI) was calculated as the difference between total binding (0) in the absence of 2 x 10-4M (-)-isoprenaline and non-specific binding(A) in the presence of the #-agonist and is expressed in pmol. The inset shows the derived Scatchard plot for specific binding. Each point is the mean of triplicate determinations, and the experiment is representative of 4 separate experiments. culated relative proportions of these were 18%: 82% value of the KD (as determined above) in order to by use of the atenolol data, and 20% : 80% by use of approach complete saturation of binding sites with the ICI 118551 data (in good agreement with the radioligand. Equilibrium binding was attained in proportions derived from the Hofstee analysis). In each case by incubating with radioligand for 45min liver preparations from newborn and one day-old as determined above (see Figure 1). Total and spe- rats the corresponding relative proportions were cific binding in the assays was shown to be directly 7: 93% (atenolol) and 10: 90% (ICI 118551) at both proportional to the amount of protein in the assay ages by use of the curve-fitting approach. tube up to 0.4mg. Saturation binding assays were carried out on rat liver preparations from animals of different foetal and postnatal ages. A typical binding Saturation binding assays ofreceptors in perinatal rat isotherm with a liver preparation from 20 day-old liver preparations foetal rats is shown in Figure 5, together with the Scatchard analysis of the data. At all ages studied, For saturation binding assays, a minimum of 10 con- non-specific binding ranged from 5-15% of the total centrations between 10 and 600pM [125I]- binding observed. Scatchard analysis of the data iodopindolol was used in each assay, the top always resulted in linear plots (mean r + s.e. mean of concentration representing an approximate 10 fold 0.99 + 0.01, P < 0.001), and Hill coefficients close to P-ADRENOCEPTORS IN PERINATAL LIVER 823

Table 1 ['251]-iodopindolol binding capacities in rat liver membrane preparations during perinatal development Bmax (fmol mg-1 protein) Adrenoceptor type Day 20 p.c. Day 21 p.c. Newborn Day 1 p.p. Day 2 p.p.

Total fl-receptors ***13.0 + 2.6 **18.3 + 1.3 30.4 ± 2.4 *21.6 + 2.4 **19.9 + 1.2 KD(PM) 66.4 + 16.0 85.8 + 4.7 81.2 + 9.7 72.9 + 10.6 69.9 + 5.2 #2-receptors ***12.3 + 2.3 **15.3 + 2.0 28.5 + 1.6 **19.8 + 1.7 *18.5 ± 3.7 KD(pM) 94.7 + 9.2 81.8 + 5.5 86.2 + 20.9 101.7 + 19.4 76.4 + 6.1

Maximal binding capacities (B..,,) were determined by saturation binding assay at 250C with 40000g particulate preparations of liver homogenates from rats of different perinatal ages. Total fi-adrenoceptor binding capacity and that attributable to apparent P2-adrenoceptor binding were determined in the absence and presence of 5 x 10-7 M atenolol, respectively. Results are given as mean values + s.e. mean for 3 or 4 different experiments (each involving triplicate determinations). Statistical analysis was by Student's t-test and values different from those in newborn rats are indicated by * P < 0.05, ** P < 0.01, *** P < 0.001. p.c., foetal age post coitum; pp., postnatal age post partum. unity (mean + s.e. mean of 1.00 + 0.10) indicating an in saturation binding assays performed with and absence of co-operative interactions. No significant without the inclusion of atenolol (Table 1); it is pos- change in KD (75 + 7 pM, mean + s.e. mean, n = 16) sible to calculate the proportion of the total f- for [1251]-iodopindolol was observed with age. In adrenoceptor population which is apparently due to contrast, B,,X values for binding capacity showed a fi1- or fl2-adrenoceptor subtypes. For the different clear age dependence (Table 1). Total f-adrenoceptor ages, the proportion of apparent fl2-adrenoceptors is binding capacity increased during late foetal life by 84-95% of total P-adrenoceptor binding. This agrees 2.3 fold from 20 days post coitum to birth, falling in with the estimates of adrenoceptor subtype propor- the first postnatal days of life to 71% and 65% at 1 tions derived from displacement curves (see above) and 2 days post partum compared to the peak value and thus provides justification for the use of atenolol found at birth. to mask fil-sites during saturation binding assays. By including 5 x 10- 7M atenolol (a concentration calculated to displace 90-100% of #1- adrenoceptor- bound ['251]-iodopindolol and < 10% of #2-specific Discussion radioligand binding at 70PM of the radioligand, see Figure 3), it was possible to conduct saturation Use of [125I]-iodopindolol to assay f-adrenoceptors binding assays which would define the apparent in perinatal rat liver fl2-adrenoceptor binding. Although pindolol is not specific for #2- adrenoceptors, and therefore at the In this study we have used the high affinity specific highest concentrations might compete to some fl-adrenoceptor antagonist (-_[1'25I]-iodopindolol extent with the Pl-adrenoceptor binding of atenolol, to characterize and quantitate fI-adrenoceptor Scatchard plots were linear and allowed calculation binding and subtype specificity. The binding of of binding affinities and maximal binding capacities [125I]-iodopindolol to perinatal rat liver membranes at the different ages (Table 1). Again there was no was rapid, saturable, reversible and showed displace- significant change in apparent KD (88 + 12pM, ment characteristics typical of binding to mean + s.e. mean, n = 16) with age, but the increase P2-adrenoceptors. The radioligand showed no selec- in apparent fl2-adrenoceptor binding capacity in late tivity for #1- or fl2-adrenoceptors. The dissociation foetal life, and its subsequent decline post partum, constant (KD) of ['251]-iodopindolol binding was paralleled the observations for total f-adrenoceptor defined both by saturation binding assay (75 pM) and binding capacity described above. Because of the by kinetic analysis of radioligand association and very high relative proportion of l2- to dissociation (61 pM). The values were in good agree- fl1-adrenoceptors in perinatal rat liver (see above), it ment with each other and with other published proved impossible to conduct competition saturation values in liver (68 pM, McMillan et al., 1983; 65 pM, binding assays for fl-adrenoceptors by using ICI Dax et al., 1986). Computer analysis by curve-fitting 118551 (at 5 x 10-8M, calculated to displace procedures (Hancock et al., 1979) and procedures 90-100% of 70pM f2-adrenoceptor-bound [12I]_ based on graphical extrapolation of Hofstee plots iodopindolol); the very low and variable radioligand (Rugg et al., 1978) derived from radioligand displace- binding and very high apparent KD values for ment studies with fl1- and fl2-subtype selective drugs, [125I]-iodopindolol contributed to the difficulties. provided evidence in both cases of binding of [125I]_ By comparing the binding capacities (B. ) obtained iodopindolol to both adrenoceptor subtypes. Using 824 K. SNELL & C.A. EVANS these data, and data derived from competition satu- conclude that [125I]-iodopindolol is the most suit- ration binding assays, we could show independently able, indeed the only, radioligand currently available that the predominant fi-adrenoceptor population of for determining f-adrenoceptor binding in rat liver, perinatal rat liver is of the ,82-subtype (80-95%). at least in the relatively crude membrane prep- Nevertheless, the evidence for a small proportion of arations necessarily employed in this work. fil-subtype binding suggests that in foetal rat liver, as in some other tissues (Dickinson et al., 1981), both Perinatal development ofrat hepatic P-adrenoceptors adrenoceptor subtypes can coexist simultaneously. [1251]-iodopindolol is a relatively recently intro- We have shown that the fl2-subtype is the predomi- duced radiolabelled antagonist for studying fi- nant (> 80%) f-adrenoceptor in perinatal rat liver at adrenoceptors (Barovsky & Brooker, 1980). While all ages studied and that the f2-adrenoceptor con- other radioligands have proved useful for assessing centration increases in late foetal liver, more than fi-adrenoceptor binding in tissues where this receptor doubling between 20 days post coitum and birth, and subtype predominates and is in relatively high con- then declines postnatally. The measurements were centration, quantitation in rat liver and other tissues carried out on whole rat liver membrane prep- has proved difficult and sometimes impossible arations and it is important in a longitudinal com- (Krawietz & Erdmann, 1979; Dax et al., 1981; 1982). parison with age that the method of preparation is We attempted to use [3H]-dihydroalprenolol to consistent and quantitative. To this end a relatively characterize f-adrenoceptors in perinatal rat liver. simple procedure was employed for membrane prep- Despite using the f-adrenoceptor agonist (-)- aration which would avoid the inevitable losses of isoprenaline to characterize specific binding (see recovery associated with more extensive purification. Nahorski & Richardson, 1979), non-specific binding The recovery of membrane protein was not signifi- was both great (usually between 60 and 70% of total cantly different between the different ages studied. binding) and highly variable. The inclusion of the a- The use of the high affinity, highly specific radioli- adrenoceptor antagonist phentolamine at 10-0M, as gand ['25I]-iodopindolol undoubtedly contributed suggested by Dax et al. (1981), while eliminating a to the accurate quantitation of f-adrenoceptors small proportion of the non-stereospecifically dis- attainable with these relatively crude membrane placeable binding, did not resolve the difficulties preparations, as noted by others (Dax et al., 1986). encountered with this radioligand. These problems By using whole liver, although purification losses were not apparent with cat ventricular membrane were minimized, the cellular heterogeneity of the preparations and KD (1.7 nM) and B.,,, values tissue was retained. This is a significant point for (28fmolmg-1 protein) comparable to those in the foetal rat liver, in which at 20 days post coitum 30% literature were obtained (Minneman & Molinoff, of the volume fraction of the liver is attributable to 1980; Dax et al., 1981; Munnich et al., 1981; Sharma haematopoietic cells (Greengard et al., 1972). Rat & Corr, 1983). We conclude that [3H]- reticulocytes and erythrocytes have a high concen- dihydroalprenolol is unsuitable for the determi- tration of fl-adrenoceptors (Nahorski, 1981; Dick- nation of P-adrenoceptor binding in rat liver, even at inson et al., 1981) which will contribute to the foetal and perinatal ages. Similar attempts using binding observed in whole liver membrane prep- ['251]- as a radioligand for char- arations. However, since the volume fraction of rat acterizing fi-adrenoceptor binding in perinatal rat liver occupied by haematopoietic cells falls from liver were also unsuccessful. Despite an acceptably 30% to 10% between 20 days post coitum and birth, low level of non-specific (non-displaceable by (-)- at the time when whole liver fi-adrenoceptor concen- isoprenaline) binding of the order of 15-30% of total tration doubles, the increase in fl-adrenoceptor con- binding, saturation binding assays gave unaccept- centration in membranes from whole liver must be ably erratic Scatchard plots. In radioligand associ- attributable to differentiation of liver parenchymal ation binding studies we observed that equilibrium cells. Indeed, for this reason, the magnitude of the binding was not attained within 60min, in agree- late foetal increase in parenchymal liver cell fi- ment with others (Brodde et al., 1981; Engel et al., adrenoceptor concentration may well be an under- 1981). The large cyano grouping on the radioligand estimate. molecule may impede its access to the binding site of The findings in the present study are apparently in the P-adrenoceptor, thus increasing the time to reach contradiction to the only other published study of equilibrium (cf. iodopindolol in the present study). the perinatal development of rat liver /3- Furthermore, Engel et al. (1981) have noted biphasic adrenoceptors, by McMillian et al. (1983). By use of dissociation characteristics (with less than 50% total ['l25]-iodopindolol binding assays, they demon- dissociation in 8 h) with this radioligand, in contrast strated an apparent fall in ,B-adrenoceptor concen- to the monophasic dissociation observed in all cases tration (assumed by them to represent solely with [125I]-iodopindolol in the present study. We fl2-subtype adrenoceptors) between 20.5 days post fi-ADRENOCEPTORS IN PERINATAL LIVER 825 coitum and 0.5 days post partum. However, because Aggerbeck et al., 1980; Morgan et al., 1983). In par- of the ages selected for study, they would not have ticular it appears that glycogenolysis in perinatal rat observed the increase in #-adrenoceptor concentra- liver is mediated by P-adrenoceptors (Sherline et al., tion in the last two days of gestation demonstrated 1974; Moncany & Plas, 1980; Huhn et al., 1983). We in the present study. There is agreement on the post- have found that the sensitivity of hepatic glycoge- natal decline of fl-adrenoceptor concentration in rat nolysis to fi-adrenoceptor activation increases in late liver and McMillian et al. (1983) show that this foetal life to reach a peak at birth (unpublished decline continues into adult life, and has been docu- observations) in a manner analogous to that mented for /3-adrenoceptor-mediated responses in rat described here for #2-adrenoceptor concentration in liver (Blair et al., 1979; Morgan et al., 1983). liver membranes. This strongly suggests that the It is generally accepted that in the adult male rat present observations have a physiological relevance liver adrenergic responses are mediated by for the development of hepatic adrenergic respon- a1-adrenoceptors (see Exton, 1979; Kunos, 1984). siveness, but does not exclude the possibility that Although, a-adrenoceptors have been identified in signal transduction and post-receptor mechanisms perinatal rat liver by radioligand binding studies, might also contribute to this developmental adapta- their concentration decreases between late-foetal and tion. early postnatal life (Butlen et al., 1980; McMillian et al., 1983). Indeed, according to McMillian et al. (1983) the predominant in late rat subtype foetal C.A.E. was supported by an S.E.R.C., C.A.S.E. studentship liver is the a2-adrenoceptor, although this point is in collaboration with Glaxo Group Research (Ware) Ltd. still controversial (Butlen et al., 1980). In fact, the We are grateful for generous financial support from Glaxo evidence in juvenile and neonatal rats indicates that Group Research Ltd., and for valuable discussions with Dr adrenergic responses are predominantly mediated by L.E. Martin, Dr J. Hare, and other colleagues of the Bio- P-adrenoceptors (Exton, 1979; Blair et al., 1979; chemical Pharmacology Department.

References

AGGERBECK, M., GUELLAEN, G. & HANOUNE, J. (1980). inhibitor which causes 50% inhibition (150) of an enzy- Adrenergic receptor of the a,-subtype mediates the acti- matic reaction. Biochem. Pharmacol., 22, 3099-3108. vation of the glycogen phosphorylase in normal rat CUEZVA, J.M., BURKETT, E.S., KERR, D.S., RODMAN, H.M. liver. Biochem. Pharmacol., 29, 643-645. & PATEL, M.S. (1982). The newborn of diabetic rat. I. BAROVSKY, K. & BROOKER, G. (1980) ['251]-Iodopindolol, Hormonal and metabolic changes in the postnatal a new highly selective radioiodinated fi-adrenergic period. Pediatr. Res., 16, 632-637. receptor antagonist: measurement of f-adrenergic DAVIS, D.J., DATTEL, B.J., BALLARD, P.L. & ROBERTS, J.M. receptors on intact astrocytoma cells. J. Cyclic Nucleo- (1987). a-Adrenergic receptors and cyclic adenosine tide Res., 6, 297-307. monophosphate generation in human fetal lung. BLAIR, J.B., JAMES, M.E. & FOSTER, J.L. (1979). Adrenergic Pediatr. Res., 21, 142-147. control of glucose output and adenosine 3',5'- DAX, E.M., PARTILLA, J.S. & GREGERMAN, R.I. (1981). monophosphate levels in hepatocytes from juvenile and Quantitation of beta-receptors in rat liver: confounding adult rats. J. Biol Chem., 254, 7579-7584. effect of displaceable but non-stereospecific antagonist BLAZQUEZ, E., RUBALCAVA, B., MONTESANO, R., ORCI, L. binding. J. Receptor Res., 2, 267-283. & UNGER, R.H. (1976). Development of insulin and glu- DAX, E.M., PARTILLA, J.S. & GREGERMAN, R.I. (1982). cacon binding and the adenylate cyclase response in (-)[3H]-Dihydroalprenolol binding to rat adipocyte liver membranes of the prenatal, postnatal and adult membranes: an explanation of curvilinear Scatchard rat: evidence of glucagon 'resistance'. Endocrinology, 98, plots and implications for quantitation of fi-adrenergic 1014-1023. sites. J. Lipid Res., 23, 1001-1010. BRODDE, O.-E., ENGEL, G., HOYER, D., BOCK, K.D. & DAX, E.M., PARTILLA, J.S., JACKSON, K.L. & GREGERMAN, WEBER, F. (1981). The fJ-adrenergic receptor in human R.I. (1986). [1251]-Iodopindolol binding to fi-adrenergic lymphocytes: subclassification by the use of a new receptors in tissues of the rat with particular reference radioligand, (±)-125iodocyanopindolol. Life Sci., 29, to the characterisation in liver. J. Recept. Res., 6, 165- 2189-2198. 182. BUTLEN, D., GUILLON, C., CANTAU, B. & JARD, S. (1980). DICKINSON, K., RICHARDSON, A. & NAHORSKI, S.R. Comparison of the developmental patterns of vasop- (1981). Homogeneity of beta2-adrenoceptors on rat ressin, glucagon and a-adrenergic receptors from rat erythrocytes and reticulocytes: a comparison with het- liver membranes. Mol. Cell Endocrinol., 19, 275-289. erogenous rat lung beta-adrenoceptors. Mol. Phar- CHENG, Y. & PRUSOFF, W.F. (1973). Relationship between macol., 19, 194-204. the inhibition constant (K;) and the concentration of ENGEL, G., HOYER, D., BERTHOLD, R. & WAGNER, H. 826 K. SNELL & C.A. EVANS

(1981). [l25lodo]cyanopindolol, a new ligand for Ii- AMP levels by al- and fi2-adrenergic receptors in male adrenoceptors: identification and quantitation of sub- rats. J. Biol. Chem., 258, 5103-5109. classes of fi-adrenoceptors in guinea pig. MUNNICH, A., GEYNET, P., SCHMELCK, P.H. & HANOUNE, Naunyn-Schmiederberg's Arch. Pharmacol., 317, 277- J. (1981). Rat liver, /i-adrenergic receptors: identification 285. and characterisation with (-)[3H]-dihydroalprenolol. EXTON, J.H. (1979). Mechanisms involved in ae-adrenergic Horm. Metab. Res., 13, 18-21. effects of catecholamines on liver metabolism. J. Cyclic NAHORSKI, S.R. (1981). Identification and significance of Nucleotide Res., 5, 277-287. beta-adrenoceptor subtypes. In Towards Understanding GREENGARD, O., FEDERMAN, M. & KNOX, W.E. (1972). Receptors, ed. Lamble, J.W., pp. 71-77, Amsterdam: Cytomorphometry of developing rat liver and its appli- Elsevier. cation to enzymatic differentiation. J. Cell Biol., 52, NAHORSKI, S.R. & RICHARDSON, A. (1979). Pitfalls in the 261-272. assessment of the specific binding of (-)[3H]- HANCOCK, A.A., DE LEAN, A.L. & LEFKOWITZ, R.J. (1979). dihydroalprenolol to f-adrenoceptors. Br. J. Phar- Quantitative resolution of beta-adrenergic subtypes of macol., 66, 469-470P. selective ligand binding: application of a computerized NAKAMURA, T., TOMOMURA, A., NODA, C., SHIMOJI, M. & model fitting technique. Mol. Pharmacol., 16, 1-9. ICHIHARA, A. (1983). Acquisition of a fi-adrenergic HOHN, W., SCHULZE, H.-P. & DARGEL, R. (1983). Effect of response by adult rat hepatocytes during primary glucagon, phenylephrine, and isoproterenol on glycoge- culture. J. Biol. Chem., 258, 9283-9289. nolysis and glucose release from fetal rat hepatocytes in RUGG, E.L., BARNETT, D.B. & NAHORSKI, S.R. (1978). suspension. Biol. Neonate, 44, 153-157. Coexistence of beta, and beta2 adrenoceptors in mam- KRAWIETZ, W. & ERDMANN, E. (1979). Specific and malian lung: evidence from direct binding studies. Mol. unspecific binding of (-)[3H]-dihydroalprenolol to Pharmacol., 14, 996-1005. cardiac tissue. Biochem. Pharmacol., 28, 1283-1288. SHARMA, A.D. & CORR, D.B. (1983). Modulation by amio- KUNOS, G. (1984). The hepatic ac-adrenoceptor. Trends darone of cardiac adrenergic receptors and their electro- Pharmacol. Sci., 5, 380-383. physiologic responsivity to catecholamines. Circulation, KUNOS, G., HIRATA, F., ISHAC, E.J.N. & TCHAKAROV, L. 4,99. (1984). Time-dependent conversion of ;l- to ,B- SHERLINE, P., EISEN, H. & GLINSMANN, W. (1974). Acute adrenoceptor-mediated glycogenolysis in isolated rat hormonal regulation of cyclic AMP content and glyco- liver cells: role of membrane phospholipase A2. Proc. gen phosphorylase activity in fetal liver in organ Natl. Acad. Sci., 81, 6178-6182. culture. Endocrinology, 94, 935-939. McMILLIAN, M.K., SCHANBERG, S.M. & KUHN, C.M. SNELL, K. & WALKER, D.G. (1978). Glucose metabolism in (1983). Ontogeny of rat hepatic adrenoceptors. J. Phar- the newborn rat: the role of insulin. Diabetologia, 14, macol. Exp. Ther., 227, 181-186. 59-64. MINNEMAN, K.P. & MOLINOFF, P.B. (1980). Classification SPERLING, M.A., GANGULI, S., LESLIE, N. & LANDT, K. and quantitation of ,B-adrenergic receptor subtypes. (1984). Fetal-perinatal catecholamine secretion: role in Biochem. Pharmacol., 29, 1317-1323. perinatal glucose homeostasis. Am. J. Physiol., 247, MONCANY, M.L.J. & PLAS, C. (1980). Interaction of gluca- E69-E74. gon and epinephrine in the regulation of adenosine 3',5'- WILLIAMS, L.T. & LEFKOWITZ, R.J. (1978). Receptor monophosphate-dependent glycogenolysis in the Binding Studies in Adrenergic Pharmacology. New York: cultured fetal hepatocyte. Endocrinology, 107, 1667- Raven Press. 1675. MORGAN, N.G., BLACKMORE, P.F. & EXTON, J.H. (1983). (Received October 20, 1987 Age-related changes in the control of hepatic cyclic Accepted November 2, 1987)