862 BENDECK AND NOGUCHI

25. Raul F, Noriega R, Ngi-Emvo E, Doffoel M, Grenier JF 1983 Lactase activity corticosteroids on activity of a-glucosidases in intestine of the infant rat. J is under hormonal control in the intestine of adult rat. Gut 24:648-652 Clin Invest 5 1: 1244-1 250 26. Koldovsky 0. Herbst JJ, Burke J. Sunshine P 1970 RN.4 and DNA in intestinal 30. Solimanc G, Burges EA, Levin I3 1967 Protein-calorie malnutrition: effect of mucosa during developn~entof normal and cortisone-treated rats. Growth deficient diets on enzyme levels ofjejunal mucosa of rats Br J Nutr 2155- 34:359-367 68 27. Hatch TF, Lebenthal E. Branski D 1979 Effect of early postnatal acquired 3 1. Nsi-Enivo E, Raul F 1984 Stimulation of lactase synthesis induced by starvation malnutrition on intestinal growth, disaccharidases and cntcrokinase. J Nutr in the jejunum of adult rat. Enzyme 31:45-49 109: 1874- 1879 32. Scott 1, Batt RM, Maddison YE, Peters J 198 1 Differential effect of glucocor- 28. Lebenthal E. Sunshine P. Krctchmer N 1973 Effect of prolonged nursing on ticoids on structure and function of aduit rat jejunum. Am J Physiol the activity of intestinal lactase in rats. Gastroenterology 63: 1136-1 141 241:G306-.C3 12 29. Lebenthal E. Sunshine P, Kretchmer N 1972 Effect of carbohydrate and

003 1-3998/85/1908-0862$02.00/0 PEDIATRIC RESEARCH Vol. 19, No. 8. 1985 Copyright O 1985 International Pediatric Research Foundation. Inc Prinrcd in U.S.A.

Age-Related Changes in the Control of Glycogenolysis in Rat Liver: The Significance of Changes in Receptor Density

JOHN L. BENDECK AND AKIHIKO NOGIJCHI Pediutric Re~eurcliInstit~ilr. Curdinill Giennon Children's Hospiiul, Department oJPediutrics, St. Louis University, Suini Louis, A4is.sol~ri63104

ABSTRACT. The present study examined the develop- Abbreviations mental changes in the adrenergic control of glycogenolysis in the rat model. A relatively new P-adrenergic radioligand, CAMP, 3':5'-cyclic adenosine monophosphate '251-iodocyanopindolol (ICP), was examined in binding as- GFP, guanosine triphosphate says with rat liver plasma membrane (LPM). ICP dem- Gpp(NH)p, guanylyl-imidodiphosphate onstrated both a higher specificity and a greater affinity ICP, '251-iodocyanopindolol for P-adrenergic receptors than any previously available P- LPM, liver plasma membrane adrenergic radioligand used to study rat LPM. Utilizing Kd. dissociation constant this new ligand it was found that P- density decreased from 114 + 4 fmol mg-' in newborn LPM to 19 +. 3 fmol mg-' in adult male LPM. In contrast a-adrenergic receptor density examined using 3H- The mechanism of action of epinephrine on liver glycogeno- increased from 161 -c 14 fmol mg-' in the newborn to 554 iysis varies depending on a number of factors. In 59 species ' fmo' mg-' in the ma'e' The of ICP such as the guinea pig and rabbit epinephrine-induced glycogen- displacenient assays employing various P-adrenergic olysis is mediated by the @-adrenergicreceptor system (1,2) while agonsists and antagonists indicated that ICP binding sites in the adult male rat the system predominates (3, were P2-adrenergic receptors' Both triphosphate 4). In the adult female rat glycogenolysis has recently been shown and its nonhydrolyzable synthetic analog guanylyl-imido- to be under the influence of both a- and P-adrenergic stimuli (4). di~hOs~hatelowered the for ICP Several recent studies have also the impoflance of binding sites similarly in newborn and adult LPM. Thus the age of the experimental animals on the adrenergic control of the of receptor to guanine nucleotide g~ycogeno~ysis~Inthese investigations it was demonstrated that protein appeared to be same in both age groups ex- P-adrenergic stimuli resulted in a marked rise in CAMPlevels in amined. In isolated hepatocytes glycogen phosphorylase hepatocytes from juvenile rats but little rise was noted in the activation was mediated by B2-adrenergic stimi~liin the hepatocytes from adult male rats. They also showed that glucose and a-adrenergic in the release from the cells occurred following both a- and 0-adrenergic adult male. These results suggest that the change in gly- stimuli in the juvenile hepatocytes but only following a-adrener- cogen phosphorylase activation from P- to predominantly gic stimulation in the adult hepatocytes (5. 6). In another study a-adrenergic mechanisms seen with maturation is related Using cultured fetal hepalocyles and gly- to changes in receptor density- (Pediar Res 19:862--868, 1985 cogeIlo~ysiswere mediated solely by padrenergic (7). However, the results of this study must be questioned since it has recently been reported that the culturing of adult male Received October 29, 1984: accepted April 3, 1985. hepatocytes results in an increase in P-adrenergic receptor density Reprint requests John L. Bendeck, M.D., Cardinal Glennon Children's Hospital, and a in adrellergic mediated gl~cOgenO1~sis a 1465 South Grand Boulevard, St. Louis, MO 63 104. to @ control (8). Nonetheless, several of the latter studies seem ADRENERGIC CONTROL OF GLYCOGENOLYSIS 863 to suggest that in rat liver the adrenergic control of glycogenolysis assays. Under these conditions, the protein was stable for bindin;: undergoes developmental changes; and it has been speculated studies for at least 2 months. that this maturational change from P- to predomina~ltlya- Assay of '251-ICPbinding. Binding assays were camed out in adrenergic control may be related to changes in the number or duplicate or triplicate at 37" C by incubating membrane (20-80 more specifically density of adrenergic plasma membrane recep- pg) in a final volume of 250 p1 of 10-50 mM Mg-Tris buffer (pH tors. However, initial attempts by us and others (8) to character- 7.4) and ICP and/or competing drugs for 1 h. For ICP saturation ize adequately the p-adrenergic receptor in rat LPM have been studies the membrane protein was incubated with increasing unsuccessful utilizing '251-iodohydroxybenzylpindololbecause of final concentrations of ICP ranging from 20-300 pM. For ICI' its high nonspecific binding. In the present study we utilized a displacement studies membrane protein and ICP (30 pM) were new 6-adrenergic radioligand, ICP, in binding assays to deter- incubated with increasing concentrations of the competing li- mine 0-adrenergic receptor density in purified plasma mem- gand. Specific concentrations are noted in the legends of thc branes from newborn and adult male rats because at least in appropriate figures. Reactions were terminated by the addition other species and tissues it has been shown to have a greater of 4 ml of ice cold Mg-Tris buffer and bound and free ligantl affinity and specificity for the P-adrenergic receptor than any were separated by rapid filtration through Whatman GF/B glass previously available radioligands (9). a-Adrenergic receptor den- fiber filters (Whatman, Inc., Clifton, NJ) which were then washed sity was also investigated in the same membrane preparations with an additional 12 ml of the buffer. The filter discs were using 3H-prazosin. The results of binding assays on plasma allowed to completely dry before the radioactivity was measured membranes was then correlated with adrenergic stimulation of in a -y counter at 80% efficiency (United Technologies Packard glycogen phosphorylase in isolated hepatocytes. Our results dem- Auto-Gamma 500). Specific binding of the ICP was defined as onstrate that with maturation o-adrenergic receptor density de- the amount of the label bound in the absence of competing creases while a-adrenergic receptor density increases. In addition iigand minus the amount bound in the presence of M (-)- the adrenergic stimulation of glycogen phosphorylase in isolated . Under the conditions employed specific binding was hepatocytes was mediated solely by the o-adrenergic system in a linear function of the protein concentration and was consist- the newborn and predominantly by the a-adrenergic system in ently greater than 80% of total binding. the adult male. These findings support the hypothesis that Assaj~of 3H-prazosin binding. Binding assays were camed out changes in plasma membrane receptor density are responsible in duplicate at 25" C by incubating membrane (20-80 ug) in a for the maturational changes in the adrenergic control of glyco- final volume of 1 ml of 10-50 mM Mg-Tris buffer (pH 7.4) anti genolysis. We also were able to show that ICP binding sites are 'H-prazosin at a final concentration of 50-600 pM for 30 min. f12 subtype adrenergic receptors. Termination of the assay and separation of bound from free ligand was done as described above for ICP. The dried discs were MATERIALS AND METHODS placed in 7 ml of scintillation fluid in glass vials and the radio- activity measured in a beta counter (United Technologies Pack- Plasma Membrane preparation. Purified plasma membranes ard, Tri-Carb 460). Specific binding was defined as previous1:y were prepared from the livers of Sprague-Dawley rats obtained mentioned using M(+)- as the competing from Sasco Inc. (St. Louis, MO) by the method of Neville (10) ligand and was consistently greater than 70% of total binding. with slight modifications of several of the centrifugation steps. Nepatocytepreparatioiz. isolated hepatocytes were obtained by Animals were sacrificed by intraperitoneal pentobarbital in adults in vivo perfusion of the livers with a collagenase-containin:g and decapitation in newborns. Pentobarbital did not effect recep- Krebs-Ringer bicarbonate buffer pH 7.4. In the adult rats thle tor density or Kd in preliminary investigations in adult rats. The livers were perfused according to the method of Berry (13). In removed livers were inlmediately placed in ice cold 1.0 mM the newborn rats in which the hepatocytes from two animals NaHC03 buffer. In the adult males, which were older than 60 were pooled the method of Ferri et al. (14) was used. In this case days and weighed 195-265 g, 10 g of liver tissue from each the perfusate enters the liver via a 23-gauge butterfly needlse animal was used in the preparation of each membrane. In the introduced into the inferior vena cava above the renal veins and newborns aged 5-7 days weighing 12- 16 g each the livers of eight exits via the severed portal vein. Gephalad flow of perfusate to nine different animals from a given litter were pooled (4-6 g above the liver was avoided by ligation of the inferior vena cav,a total) in preparing each membrane. After weighing the livers below the heart. Collagenase (35 mg/100 ml) and 0.102 M CaC12 were minced in 19 volumes of 1.0 mM NaHC03 buffer and ( 1.14 m1/ 100 ml) were added to the buffer only after the exiting homogenized in a Dounce homogenizer by eight strokes with perfusate was grossly free of blood and then the perfusion was Pestle A. Each homogenate was filtered through one layer each allowed to continue for another 20-30 min during which time of gauze and cheesecloth; and after removing samples for protein the perfusate was continually gassed with a 95%:5% 02:C02 and 5'-nucleotidase assays, 1.0 mM NaHCO, was added to a mixture. The livers were then gently removed into a beaker final volume of 250 ml which was well mixed and then centri- containing the perfusion media and gently teased to release fuged for 20 min at 3500 x g. The pellets were added to isolated hepatocytes. After filtering through cheesecloth and cen- approximately 11 ml of 69% (w/w) sucrose and then adjusted to trifugation at 60 x g for 30 s to separate the parenchymal cells a final sucrose concentration of 44% by the addition of either the pellet was resuspended in perfusion media in a flask into more 69% sucrose or buffer solution. Fifteen to twenty milliliters which a 95%:5% 02:C02mixture was continuously introducedl. of 42.3% (w/w) sucrose was carefully layered over the resus- The cells were then placed in a 37" C AquaTherm Water Bat11 pended pellets and the samples centrifuged at 4" C in a Beckman Shaker (New Brunswick Scientific Company, Inc., Edison, NJ) SW 27 roter for 2 h at 90,000 x g. The membrane which floated at 100 rev/min for 60-90 min before being used. Viability of the on top of the 42.3% sucrose layer were removed, mixed gently hepatocytes was checked by Trypan blue exclusion irnmediate1.y in 25 ml of the buffer solution, and centrifuged at 15,000 X g before and after stimulation studies and was routinely greater for 20 min. The pellets were then resuspended in 3-5 ml of the than 90%. Cell concentration was 2-6 x lo6 cells/ml and con- buffer which yielded a final protein concentration of 2-6 mg/ml tamination by nonparenchymal cells was consistently less than as determined by the method of Lowry et al. (1 1) using bovine 10% as determined by light microscopy. serum albumin as standard. Purification of the samples was Phosphorj?lasea dose response assay. For the adult assays 5001- assessed by determining protein recovery and 5'-nucleotidase pl aliquots of the hepatocytes were incubated with either adre- activity as described by Wendell and linkeless (12). Once pre- nergic agonists or agonists plus antagonists at 37" C in the water pared, membranes were immediately snap frozen in an acetone- bath shaker. Stimulation was stopped at exactly 2 min by re- dry ice bath and then stored at -60" C until used in binding moving 200 11 aliquots of the mixture into test tubes on ice as 864 BEWDECK AND NOGUCHI

described by Hutson et a/.(1 5). This period of time was chosen and to evaluate ICP binding to rat liver plasma membrane because in preliminary studies hormonal stirnuladion was maxi- preliminary studies were undertaken. Figure 1 describes the mal at 2 min in both raewborn and adult hepatocytes. The cells association-dissociation reaction of ICP with liver plasma mem- were then homogenized for 10 seconds at half speeed on an brane protein. It was found that equilibrium was reached within Ultra-Turrax homogenizer (Tekmar Company, Cincinnati, OH). 40 rnin and although not shown in Figure P the amount of ICP Newborn assays were performed identically except half volume bound remained the same beyond this point out until 4 h time. aliquots were used. Phosphorylase a activity was then determined In this particular experiment the 0-adrenegic antagonist (-)- in the stimulated hepatocytes by measuring the incorporation of alprenolol was added to a final concentration of 10-' M at 40 I4C-G-1-P into glycogen by the method of Gilboe et a/.(16). min to examine the dissociation reaction. Insets A and B show Mlerials. (-)-Epinephrine, (-)-, I-)-isopro- logarithmic transformations of the specific binding data for the terenol, (-)-alprenolol, and guanosine triphosphate were ob- association and dissociation reactions respectively. tained from Sigma Chemical, St. Louis, MO; (-)-metroprolo1 In order to determine if altering the protein concentration and jk)-phentolamine were from Ciba Pharmaceutical, Summit, would affect ICP binding, 80 pM final concentrations of the NJ: '2SI-iodocyanopindololarid H-prazosin were obtained from radioligand were incubated with various concentrations of mem- New England Nuclear, Boston, MA; 14C-glmcose-l-ph~sphale brane protein ranging from 0.04-0.32 mg/ml for 1 h at 37" C. from Arnersham International Ltd., Arnersham, UK; guanylyl- The results which are indicated in Figure 2 demonstrate that imidodiphosphate was obtained from Boerhinger Mannheim, under the conditions employed specific binding of IGP to the New York, NY; (&)-epinephrine from Sterling Winthrop Re- membrane is directly proportional to protein concentration and search Institute, Rensselaer, NY: (-)- from Bristol Myers represents greater than 85% of total binding. Company, Evansville, IN; and collagenase, type I from Wor- thington Pharmaceutical, Freehold, NJ.

RESULTS Purification of the membranes was assessed by measwing protein recovery and 5'-nucleotidase activity and the results are presented in Table I. The findings suggest that while both adult and newborn LPM were highly purified the degree of purification was slightly higher in the adult membrane preparations. In order to determ~neappropriate conditions for binding assays

Table 1. Mrmhrune aurificntion* Newborn Adult .04 .88 .12 .I6 .20 .24 .28 .32 .36 Protein in crude homogenate (mg 135 ? 8 mg/ml 162 f 16 mg/ml [Protein] mglml protein per g wet liver) Fig. 2. PCP binding as a function of protein concentration. Eighty % protein recovery (crude ho- 2.6 It 0.7% 1.3 + 0.4% pM concentrations of ICP were incubated with protein concentrations rnogenatc to LPM) ranging from 0.04 to 0.36 rnglrnl for 1 h at 37" C in the absence (a total 5'-Nuclcotidase activity increase 7 ? 1 times 1 1 + 2 drmes binding) and presence of M (-)-alprenolo1 (A nonspecific binding) (LPM vi crude homogenate) for the determination of specific binding (0).Data points are the means * Results represent the mean -+ SE of five membrane preparations. of triplicate determinations.

IQ V 1- u'5 15 2b 60 12Q 180 L Minutes Minutes "0 410 6b 810 Ibo 1:o i;jo 1;3o 2bo 2L0 Minutes Fig. I. Forward and reverse kinetics of ICP binding to newborn LPM. Membrane (0.2 mg/ml) and ligand (30 pM) were incubated at 37" C; and at 40 rnin (-)-alprenolo1 was added to a final concentration of M to initiate the dissociation reaction. At the times indicated 250-pl aliqusts were rcrnoved in duplicate and assayed for bound ICP. B, represents amount of ICP bound at the given times while B, represents the amount bound at equilibrium. The inset (A)is a logarithmic transformation of the binding data of the initial phase of the association reaction where (Ro) and (Lo) are unoccupied reccpror and free Bigand concentrations at the beginning of the readion and 1x1 is the concentration of bound Pigand at time t. The association rate constant is determined from the slope of this line by the following equation Kon = slope (2.303)/[(Lo)-(Ro)]. The inset (B) shows In(1CYP) bound vcr:i-~tstime. By nonlinear regression analysis K-, and K-* were determined (see text). ADRENERCIC CONTROL. OF C;LYCOGENOLYSIS 865

In another study it was found that the addition of (?)-phen- 8-adrenergic radioligands (17, 18), under the conditions em- tolamine at a final concentration of M had no affect on ployed in our binding assays ICP does not bind to a-adrenergic either specific or nonspecific binding of ICP to membrane (data receptors to any appreciable degree. not presented). This indicates that, unlike some other iodinated Further characterization of the ICP binding sites was deter- mined by a number of displacement studies. As shown in Figure 3 the order of potency for displacing ICP from the membrane

w (-) lsoproterenol preparations was isoproterenol > epinephrine > norepinephrine c---o (-) Epinephrine which is typical for p2-adrenergic receptors. The data in Figure 3 A-A (-) Norepinephrine also demonstrates that ICP is stereoselectively displaced by the A-A (+) Epinephrine (-) and (+) forms of epinephrine. That ICP binding sites are P2-adrenergic receptors was further deduced by other displacement studies using subtype selective and nonselective 8-adrenergic ligands (Fig. 4). It was found that alprenolol, a nonspecific 8-. was more ef- fective in displacing ICP than zinterol, a P2-, and approximately three orders of magnitude more effective than , a 8,-adrenergic antagonist. Inhibition of ICP binding by epinephrine was also investigated in the absence and presence of GTP and its nonhydrolyzable I09876543 synthetic analog, Gpp(NH)p. The addition of GTP resulted in a -log [MI shift of the right in the ICP displacement curve (data not shown) Fig. 3. Inhibition of ICP binding to ncwborn LPM by classical adre- and an increase in the Hill coefficient from 0.46 to 0.7 1. Similar ncrgic agonists. Tuenty to thi11y pg of membrane protein was incubated studies were also carried out using Gpp(NHp) in both adult and wit11 ICP (30 pM) and concentrations of competing ligands ranging from newborn membrane preparations (Fig. 5). In both the addition 10-" to lo-' M for 1 11 at 37" C. Data points are the mean of three of the GTP analog resulted in a similar shift to the right in thc cxpcrimcnts cach done in triplicate. displacement curve and increase in the Hill coefficient as was seen with GTP. Table 2 summarizes the data obtained from ICP and 'H-prazosin saturation binding studies. Kd and receptor density (B,,,) were determined from Scatchard plots of specific binding data, representative examples of which are shown in Figure 6. ICP (badrenergic) binding sites declined significantly with age (1 14 k 4 fmol mg-I in the newborns ver:szis 19 ? 3 fmol mg-I in the adult) while 'H-prazosin (a-adrenergic) binding sites in-

Table 2. Surnmury ofreceptor binding in nebvhorn und ud~rlt L,Phl 125 I- 3H-prazosin iodocyanopindolol

Kd Bmax 16 Bmax " 109876543

n Wt (g) (pmol)~ (fmol/mg)* (pmol) (finol/mg)* -log [MI -- Newborn 5 12-16 30k2 1143~4 103f7 161*14 Fig. 4. Inhibition of ICP binding to newborn LPM by p-adrenergic Adult 5 195-265 31f5 I9k3t 58+11 554159$ ligands. Twenty to thirty pg of membrane protein was incubated with ICP (30 pM) and concentrations of the competing ligand ranging from * fmol bound per mg purified LPM. lo-' to lo-' M for I h at 37" C. Data points arc the mean of three t Adult < newborn 11 < 0.001. experiments each done in duplicate. $ Adult > newborn p < 0.001 by Student's / test.

Adult Newborn

H Epinephrine H Epinephrine 100 - 100 - - Epinephrine - Epinephrine 80 - +~pp(~~)p(1o-~M) 80 - K C .-0 .-0 .t: 60 - G.= 60- L2 c .c c 5 40- - 40- 8 s 20 - 20 -

T Y I I I I I r I I I I I I 9876543 9876543 -log [Epi] -log [Epi] Fig. 5. Inhibition of ICI' binding to newborn and adult LPM by epinephrine in the presence and absence of GPP(NH)p. Twenty to thirty pg of membrane protein was incubated in tlie presence (0)and absence (0)of Gpp(NH)p M) for 1 h at 37" C. Data points are the mean of two to three expcrirnents each done in dupllcatc or triplicate. BENDECK AND NOGUCHI

B. Adult

40 i

A,. Newborn 120 1

Fig. 6. Newborn and adult ICP saturation binding curves and Scatchard plots of specific binding data. Membrane protein (0.2-0.3 mg/ml) was incubated with incrcasing final conccntrations of ICP ranging from 20-300 pM for I h at 37" C in the absence (0 total binding) and presence of lo-% (-)-alprenolol (A nonspecific binding) for the determination of specific binding (0).Data points are the mean of triplicate determination at each ligand concentration. B/F. bound to free ligand concentration.

(-) lsoproternol Epi (10-5M) + Phe (3*10-5M) (-) Epinephrine Epi (1CW) + Alp (3*1C5M) I

I I I I 1 I I 5 Day Adult Fig. 8. a and P-Adrenergic stimulation of glycogen phosphorylase in Fig. 7. Newborn glycogen phosphorylase dose response. Aliquots, 250 newborn and adult hepatocytes. Isolated hepatocytes from 5-day and F1, of isolated hepatocytes from 5- to 7-day-old rats were stimulated with adult rats were stimulated with (-)-epinephrine (lo-' M) and either (?)- cithcr (-)-isoproterenol (0. (-)-epinephrine (0), or (-)-norepinephrine phentolamine (3 x lo-' M) to represent P-adrenergic stimulation or (-)- (A) in final conccntrations ranging from lo-" to lo-' M for 2 min at alprenolol(3 x M) to represent a-adrenergic stimulation for 2 min 37" C. Results are exprcsscd as percentage of maximum phosphorylase at 37" C. Results are expressed as percentage of basal phosphorylase a (1 activity ~w.sll.sagonist concentration. Data points are means of four to activity in the absence of stimulation. The data points are the mean + five separatc experiments SE of six to eight experiments. * 5 day > adult p < 0.01 0 adult > 5 day p < 0.0 I by Student's r test. creased significantly (16 1 f 14 fmol mg-' in the newborn versus hepatocytes P-adrenergic stimulation resulted in an almost 100% 554 2 59 fmol mg-' in the adult). The Kd value ofapproximately 30 pM for ICP represents an affinitythree to four times greater increase above basal in phosphorylase a activity but a-adrenergic than any previously available iodinated P-adrenergic radioligand stimulation was without effect. In the adult male hepatocytes a significant rise in phosphorylase u activity was only seen with (9). The glycogenolytic response of newborn hepatocytes to adre- a-adrenergic stimulation. nergic stimulation was assessed by measuring phosphorylase u DISCUSSION activity and the results are presented in Figure 7. The order of potency for stimulating glycogen phosphorylase was isoproter- Attempts to characterize adequately the 8-adrenergic receptor en01 > cpinephrine > norepinephrine which is characteristic of in newborn rat purified LPM have been unsuccessful using B7-adrenergic responsiveness. In another series of experiments previously available radioligands such as 1251-iodohydroxyben- the relative contributions of cu- and P-adrenergic stimulation on zylpindolol because of its high nonspecific binding (8). This glycogen phospholylase in both newborn and adult hepatocytes radioligand has been reported to bind to sites in rat was examined (Fig. 8). Hepatocytes were stimulated with a cerebral cortex (19)and much of its nonspecific binding can be combination of (-)-epinephrine (lo--' M) and either (+)-phen- eliminated in rat glioma cells by the addition of phentolamine, tolamine (3 x M) or (-)-alprenolo1 (3 x lo-' M) to represent an a-adrenergic antagonist (17, 18). In the present study we either 8- or cu-adrenergic stimulation, respectively. In the 5-day utilized a relatively new radioligand, ICP. Under the conditions ADRENERGIC CONTROL OF GLYCOGENOLYSIS 867 employed in our assays this ligand demonstrated rapid, reversi- norepinephrine, or locally released norepinephrine upon sym- ble, and stereoselective binding as well as a very high affinity pathetic nerve stimulation. Innervation of the rat liver is re- (Kd of 30 pM) and specificity for @-adrenergicreceptors. The stricted to primarily vessels in the portal space and hilus (23, 24) biphasic nature and shallowness of the ICP dissociation reaction as opposed to guinea pig, cat, primates, and humans where with the addition of antagonist have been observed elsewhere adrenergic nerves penetrate deep into the parenchyma (23-25). (9). The calculations of the dissociation rate constants are based However, there is evidence that glucose output from the liver is on the assumption that there are interconvertible receptor con- directly regulated by the sympathetic nervous system in the rat formations resulting in a rapidly formed ICP-receptor complex (26). The development of hepatic innervation in the rat has not which can dissociate quickly and a thermodynamically favored been reported to our knowledge and it remains to be studied ICP-receptor complex that dissociates more slowly as postulated whether the adrenergic receptor responsiveness changes are re- by Engel et al. (9) The high affinity and specificity of ICP binding lated to the developmental changes in innervation. While func- made it possible to demonstrate that there was a significant tionally unimportant in the adult rat, P-adrenergic control of decline in 8-adrenergic receptor density in LPM seen with mat- glycogenolysis in the newborn may be essential in maintaining uration of the male rat. In addition we were able to determine glucose homeostasis. As we have demonstrated, although that ICP binding sites in newborn LPM were P2-adrenergic a-adrenergic receptors are present in the newborn hepatocyte in receptors. The a-adrenergic receptor was also examined in these concentrations even greater than 0-adrenergic receptors, their same membranes and it was found that concomitant to the stimulation does not lead to a rise in glycogen phosphorylase decline in p-adrenergic receptor density there was an increase in activity. In addition sensitivity of the liver to glucagon, the other a-adrenergic receptor density as measured by 3H-prazosin bind- major hormone involved in glucose homeostasis, is apparently ing sites. To our knowledge only one other study has been less in the newborn than in the adult (27). published which specifically examined developmental changes In summary we conclude that 1) ICP appears to be a very in adrenergic receptor density in rat liver (20). P-Adrenergic useful radioligand for examining the P-adrenergic receptor in rat receptors were studied using "51- and a-adrenergic re- hepatocytes and probably other tissues and species as well, 2) rat ceptors using 3H-prazosin and 3H-. Although the liver ICP binding sites are (32-adrenergic receptors, and 3) the receptor densities in the study were much lower than ours large maturational change in adrenergic-mediated glycogen phospho- increases in a-adrenergic receptor densities and decreases in rylase activation from ,B- to predominantly a-adrenergic control p-adrenergic receptor densities with maturation were also noted. is probably related to concomitant changes in the respective The lower numbers in the report are most likely due to the fact receptor densities. that a less purified membrane preparation was employed. In addition the reported Kd value for 'H-prazosin binding was at REFERENCES least an order of magnitude larger than what we found in the I. Arinze IJ, Kawai Y 1983 Adrenergic regulation of glycogcnolysis in isolated present study and in previous studies (21). A recent study (1 1) guinea pig hepatocytes: evidence that PI-receptors mediate catecholamine employing ICP and adult male LPM of similar purity as ours stimulation of glycogenolysis. Arch Biochem Biophys 225: 196-202 but prepared by an entirely different procedure reported a 2. Yorek MA, Rufo GA, Ray PD 1980 Gluconeogenesis in rabbit liver. Biochem P-adrenergic density virtually identical to that reported here; and Biophys Acta 632:s 17-526 3. Sherline P, Lynch A, Glinsmann WH 1972 Cyclic AMP and adrencrglc although no actual data were presented they stated that the receptor control of rat liver glycogen metabolism. Endocrinology 91:680- receptor density was much higher in newborn LPM. Our studies 690. ~ on isolated hepatocytes revealed that the activation of glycogen 4. Studer RK, Borle AB 1982 Differences between male and female rats in the phosphorylase by epinephrine was mediated exclusively by P2- regulation of hepatic glycogenolysis. J Biol Chem 257:7987-7993 adrenergic mechanisms in the newborn and predominantly by 5. Blair JB, James ME, Roster JL I979 Adrenergic control of glucose output and adenosine 3', 5'-monophosphate levels in hepatocytes from juvenile and m-adrenergic mechanisms in the adult. Thus it appears that the adult rats. J Biol Chem 254:7579-7584 P-adrenergic control of glycogenolysis which develops during 6. Morgan NG, Blackmore PF, Exton JH 1983 Age-related changes in the control fetal life (7) is carried over into the neonatal period; and only of hepatic cyclic AMP levels by a, and P2-adrenergic receptors in male rats. after this time does attentuation of the P-adrenergic response and J Biol Chem 2583 103-5 109 emergence of the a-adrenergic response occur (3-6). Much data 7. Moncany ML, Plas C 1980 Interaction of glucagon and epinephrine in the have been accumulating which suggest that the low P-adrenergic receptor density in the adult male is responsible for the lack of a 8. Nakamura T, ~omom"raA; Noda C, ~himojiM, lchihara A 1983 Acquisition P-adrenergic glycogen phosphorylase activation. In the present of a P-adrenergic response by adult rat hepatocytes during primary culture. study the coupling mechanism between receptor and guanyl J Biol Chem 258:9283-9289 9. Engel G, Hoyer D, Berthold R, Wagner H 1981 (+) [12510do], a nucleotide regulatory protein seemed to be the same in both new ligand for 0-adrenoreceptors in guinea pig. Naunyn Schmeiderbergs newborn and adult membrane preparations since the addition of Arch Pharmacol 3 17:277-285 GTP and Gpp(NH)p in competition assays resulted in a shift to 10. Neville DM 1968 Isolation of an organ specific protein antigen from cell the right and an increase in the Hill coefficient of the displace- surface membrane of rat liver. Biochem Biophys Acta 154:540-552 I I. Lowry OH, Rosebrough NH, Farr AL, Randall RJ I95 1 Protein measuremcnr ment curves. Recently Nakamura ct al. (8) have demonstrated with the folin phenol reagent. J Biol Chem 193:265-275 the presence of both subunits of guanyl nucleotide regulatory 12. Wendell CC, Unkeless JC 1969 Partial purification of a lipoprotein with 5'- protein in adult male hepatocytes and found that the addition of nucleotldase activity from membranes of rat liver cells. Proc Natl Acad SCI GTP did not increase adenyl cyclase activity in response to USA 61:1050-1057 isoproterenol. We (22) and others (6) have also found that, 13. Beny MN 1974 High-yield preparation of morphologically intact isolated parenchymal cells from rat liver. Meth Enzymol 32:625-63 I although P-adrenergic stimulation produces only a small rise in 14. Ferri P, Satabin P, Manoubi LE. Callikan S, Girard J 1981 Relationship cAMP accumulation in adult male hepatocytes, glucagon pro- between ketogenesis and gluconeogenesis in isolated hepatocytes from new- duces a rise in cAMP and glycogen phosphorylase activity which born rats. Biochem J 200:429-433 is similar to that seen in the newborn. These findings when taken 15. Hutson NJ, Brumley FT, Assimacopoulos FD, Harper S, Exton JH 1976 Studies on the adrenergic activation of hepatic glucose output. Studies on together suggest that the most likely explanation for the lack of the a-adrenergic activation of phosphorylase and inactivation of glycogen P-adrenergic responsiveness in adult male hepatocytes is the snythase in isolated rat liver parenchymal cells. J Biol Chem 251:5200-5208 decreased receptor density since all of the other components of 16. Gilboe DP, Larson KL, Nutall FQ 1972 Radioactive method for the assay of the P-adrenergic-mediated glycogen phosphorylase activation glycogen phosphorylase. Anal Bochem 47:20-27 pathway are present and apparently functional. The significance 17. Terasaki WL. 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19. Dickinson KtJ. Nahorski SK. Willcocks AL 108 1 Serotonin recognition s~tcs 24. Reilly FD. McCuskey PA. McCuskey RS 1978 intrahcpatic distribution of are labcllcd In cerebral cortex b) the M-adrenoreceptor antagonist '"I- nerves in the rat. Anat Rec 191:55-68 hydroxybcnzylpindolol. Rr J Pharmacol 72:165P 25. Forssman WG, Ito S I977 Hepatocyte inncrvat~onin primates. J Ccll Biol 20. McMillian MK. Schanbcrg SM. Kuhn C'M 1983 Ontogeny of rat hepatic 74:299-3 13 adrcnorcceptors. J Pharmacol Exp Ther 227: 18 1-1 86 26. Hartman H, Beckh K. Jungermann K 1982 Direct control of glycogen metab- 2 I. Noguchi A 1983 Nor~nalontogeny of <,,adrencrgic receptor in rat liver is olisrn in the pcrfused rat liver by the syrnpathet~cinnervation. Eur J Biochem thyroid hormone dependent. Endocrinology 113572-676 123:52 1-526 22. Noguch~A. Jctt I'A. Gold .4H CAMP indcpendcnt stimulation of glycogen 27. Blazqucs E, Rubalcava B. Montesano R, Orci L, Unger RH 1976 Development pho~phorylasein newborn rat hcpatoc)tes. Am J Phgs~ol248:E560-E566 of insulin and glucagon b~ndingand thc adcnylatc cyclasc rcsponsc in liver 23. Met7 W. Forssman WG 1980 lnnervat~onof the livcr in guinea pig and rat. membranes of prenatal, postnatal, and adult rat: cvidcnce of glucagon Anat Embryo1 l60:239-252 "resistance." Endocrinology 98: 10 14- 1023

003 1 -39981851 1908-0868$02.00/0 PEDIATRIC RESEARCH Vol. 19. No. 8, 1985 Copyright a. 1985 International Pediatric Rcsearch Foundation. Inc Printed it7 C' X.4.

Differential Toxicity of RCAll (Ricin) on Rabbit Intestinal Epithelium in Relation to Postnatal Maturation'

ALLAN D OLSON. THEODORE J PYSI-IER, ALFRED0 LARROSA-HARO, AKHTAR MAHMOOD. AhD RAMOh TORRES-PINFDO C'n/~,cr..c//!c~/'Olilulroiiicr Ilcul111 S(.icwc,esCotitct., Dc;i)ur.~inen/of'1~ediutrcc.s und Depur/inr>nlofPu'arhologj., Seciion ofPc~diuir.ic,G'crclrocnto.o/0,~1~ uiid "V~~~lrili~n,Okl~/~oinu Cil!:, Ok/~/?oinu73/26

ABSTRACT. The purpose of this work was to assess the Abbreviations toxic lectin ricin (RCA,,) as a probe for the study of intestinal permeability in the developing small bowel. Je- RCAI,, ricin junal explants from suckling and adult rabbits were ex- PBS, phosphate-buffered saline posed to varying dosages of RCAll for 30 min at 25" C and then cultured in toxin-free medium. The RCA,, dose re- quired to inhibit protein synthesis during 6 h of culture increased from 0.1 pg/ml in 4-day-old rabbits to 25 pg/ml in weanling rabbits. KCA,, cytotoxicity was almost com- pletely blocked by 0.1 M lactulose in all age groups. The The early part of the suckling period in rodents is characterized kinetics of '251-RCAIIbinding to purified microvillus mem- by a high level of receptor-mediated responses in small intestinal branes were determined by incubating a fixed concentration epithelium. This has been clearly shown for the jejunal transport of membrane protein (30 pg) with increasing concentrations of breast milk iminunoglobulin (1, 2) and is also inferred from of labeled lectin (2-18 @g/ml).Binding attained saturation the high content of trophic factors in breast milk (3, 4). The with adult but not with suckling animal membranes. The surface characteristics underlying this high receptor responsive- latter yielded a curvilir~earrelationship in Scatchard plots, ness in the intestine of neonatal animals are poorly understood. suggesting either several classes of binding sites or negative A marked developmental change in surface reactivities to lectins cooperativity. RCA,, binding was confined to the delipi- has been described in the small intestine of suckling rats (5-7). dated fraction of the membranes and decreased by 42% In this animal species, the intestinal microvillus membrane from 6 days old to adult age. The extreme sensitivity of undergoes a progressive shift from sialylation to fi~cosylationof coiostral epithelium to RCA,, is probably related to the glycoproteins and glycolipids during postnatal development (7). high level of endocytosis exhibited by the immature mem- In the present work, we have examined the response of rabbit brane of suckling rabbits. The development of increasing intestinal epithelium to the toxic action of RCA,,. This toxin resistance to the toxin, and associated decrease in binding, inhibits protein synthesis in intact cells (8, 9) through a mecha- might be related to disappearance of saccharide sites in nism that requires terminal nonreducing galactosyl (or N-acetyl- productive surface receptors occurring in the derelop- galactosaminyl) residues (10) in a glycoprotein receptor- capable mental course of intestinal glycosylation. (Pediatr Res 19: of transmembrane signaling for toxin internalization (1 I). This 868-872, 1985) toxin, therefore, "mimics" the mechanism of interaction of trophic hormones (12). The RCA,, membrane receptors may be Reccivcd January 7. 1985: acccptcd March 26. 1985. part or be closely associated with cell surface components in- Rcquests for reprlnts Ramon Torrcs-Pincdo. M.D.. Professor of Pcdiati-ics. P.O. volved in cell-macromolecule and cell-cell recognition (I 3). The Box 26307. Oklahoma City. OK 73 126. Suppor-tcd b) Grant tID-13441 from the National lnstitlitc of Health probing of intestinal epithelium with RCA,, may provide insight ' 1'1-cscntcd in pat-t 211 thc annual mccting of [lie American Pcdi,itric Society and into the role of glycosylation on receptor-mediated responses in thc Socict? for Pcdlatric Kcscarch. Washington. D.C.. May 3-6. 1983 the developing small bowel.