Brain Re.wurth B~tlkiin, Printed in the USA.
036 i-9230192 $5.00 t .OO
Vol. 28. pp. 4 I l-4 15. I992
Copyrightc. 1992Pergamon PressLtd.
Norepinephrine Increases Angiotensin II Binding in Rat Brain Synaptosomes COLIN
SUMNERS
Received 15 July 1991 C. ~~?~~r~pi~~p~lr~~~t~ jn~reus~s ~n~l{~I~n~inII ~i~zdin~ in VQIhrcrin .s~~~~~~fu~~(~~t~.s. BRAIN RES BIJLL 28(3) 4 1l-4 15. 199X--Synaptosomes prepared from rat hypothalamus or brainstem contain specific binding sites for [?I-angiotensin II (All). Treatment of these synaptosomes with norepinephrine (NE) (lo-500 PM) for 60 min results in a concentration-dependent increase in [‘2SI]-AII specific binding which appears to be an increase in the number rather than the affinity of these binding sites. This etfect of NE is qualitatively similar in synaptosomes prepared from neonate (one-day-old) or adult (l40-day-old) rats. Furthermore. it is antagonized by prazosin (IO PM) but not by yohimbine (IO JIM), indicating the involvement of a,-adrenergic receptors. Finally, this effect of NE may involve activation of protein kinase C (PKC) because it is mimicked by a PKC agonist (TPA. 0.8 pM; 60 min) and is blocked by a PKC antagonist (H-7. 100 FM). These results match our previous findings on the regulation of AI1 receptors in neurons cultured from the hy~thalamus and brainstem of neonate rats and provide strong evidence for a role of this catecholamine in the modulation of brain AI1 receptors. SUMNERS,
Catecholamines
Angiotensin II receptors
Hypothalamus
Brain stem
Synaptosomes
METHOD
THE brain contains specific receptors for angiotensin II (AII). with highest densities present in the hypothalamus and brainstem (6,8,15,22). AI1 hinds at these receptors and activates neural pathways, including norepinephrine (NE) neurons, ultimately leading to increases in blood pressure and fluid intake (2,5,20.23). The regulation of brain AI1 receptors represents an important way in which the central actions of AI1 are modulated. However, this regulation is di~cult to assess in the in vivo situation due to a variety of different influences that are present, including sodium ions, mineralocorticoids, estrogens, and NE (14.24.26.28). In earlier studies we utilized in vitro primary neuronal cultures prepared from neonate (one-day-old) rat brains as a model system in which to study the regulation of neuronal AI1 receptors [see (26) for review]. We determined that there are numerous regulatory influences on neuronal AI1 receptors, including NE. For example, short-term (0.5-l .O hr) incubation of hypothalamus/hrainstem neuronal co-cultures with NE results in an increased number of [‘251]-AII specific binding sites in these cells (17). This eff‘ect of NE is mediated via ol,-adrenergic receptors and involves activation of inositol phospholipid hydrolysis and protein kinase C (PKC) hut not protein synthesis. In the present study, we determined the effects of NE on the regulation of [12SI]-AII specific binding in another in vitro system, namely synaptosomes prepared from either rat hypothalamus or brainstem. In this preparation. NE elicits increases in [“‘I]AI1 specific binding that are mediated via a,-adrenergic receptors and possibly activation of PKC with no qualitative differences between synaptosomes from neonatal and adult tissue. This data suggests a role for NE in the regulation of brain AI1 receptors.
The animals used in all experiments were male SpragueDawley rats (250-300 g), purchased from Charles River Farms, Wilmington, MA. Two rats were housed per stainless steel cage and allowed free access to water and Purina Laboratory Chow (#SOOl) in a room where both temperature (25°C) and light cycle (07.00 h to 2 I .OO h) were controlled.
Bovine serum albumin fraction V (BSA). N-2-hydroxyethylpiperazine-~-2-ethanesulfonic acid (HEPES), tris (hydroxymethyl) aminomethane (Tris), ethylenediaminetetra-acetic acid (EDTA), angiotensin II (AH), phorbol- I 2-myristate- 13 acetate (TPA). H-7 [ I-(5-isoquinolinylsulfonyl) 2-methyl-piperazine], norepinephrine HCI, prazosin HCI, yohimhine HCl, and dimethylsulfoxide (DMSO) were purchased from Sigma Chemical Co., St. Louis, MO. [‘251]-AII (specific activity 1800 @.Zi/cLg)was purchased from DuPont New England Nuclear, Boston, MA. All other chemicals were purchased from Fisher Scientific, Pittsburgh. PA.
One-day-old or 140-day-old rats were sacrificed by decapitation, and their brains were dissected free and rinsed in icecold 0.9% saline. All studies were conducted between IO:30 and 1 I:30 a.m. For each brain, the brainstem (medulla ohlongata
Requests for reprints should be addressed to Dr. C. Sumners, Department of Physiology. P.O. Box 100274. JHMHC. University of Florida, Gainesville. FI. 376 IO.
411
and pans) and a hypothalamic block containing the supraoptic, paraventricular, anterior, dorsomedial, ventromedial lateral, and posterior nuclei were dissected free from the rest of the brain and all blood vessels and pia mater removed. These areas were used for two reasons: a) They contained the highest levels of brain AI1 receptors and b) these are the areas used in our neuronal cultures, so we were able to make direct comparison with these studies. The Pz pellets, enriched in synaptosomes, were prepared from these two areas basically as described previously (18) according to the method of Sgaragli et al. (21). The brain tissues were immediately homogenized using a teflon homogenizer with five to eight strokes by hand in a cold sucrose-HEPES (0.32 M and 2 mM) buffer, pH 7.6. The homogenates were centrifuged at 1000 X R for 10 min followed by a centrifugation of the supernatants at 12,000 R for 30 min. The pellet resulting from this centrifugation is termed the PZ fraction and represents a crude svnaptosomal fraction. To determine the ]‘Z51]-AII specific binding of this fraction from one-day-old and 140-day-old rats, the hypothalamic block and brainstem pellets were suspended in the sucrose-HEPES buffer at a protein concentration of 2-4 mg/ml, and AI1 binding was performed as detailed below. For the various treatments of the synaptosomal fraction, the pellets were suspended in a small volume of sucrose-HEPES buffer (onetenth the final volume) and then suspended to a final protein concentration of 2-4 mg/ml with a Krebs-Ringer buffer (KRB), which was bubbled with oxygen prior to use ( 139 mM NaCl, 5 mM KCl, 1 mM NaH2P0,, 11 mM NaHCO, . 10 mM dextrose, and 1 mM CaCl,. pH 7.6).
200 ~1 of binding buffer (150 mM NaCl; 50 mM Tris; S mM EDTA, pH 7.2; 0.04% heat-inactivated BSA; and 0.2 nM [ “51JAII) in a m~crofuge tube with (nonsp~ific binding buffer) OF without (total binding buffer) 1 FM unlabeled AI1 for 60 min at room temperature. For saturation experiments, 0.05-3 nM [“‘II-AI1 was used in the incubation mixture. Triplicate samples were used to determine the total and nonspecific binding for each sample. The protein concentration range used during the binding assay fell on the linear region of the binding curve. Time courses of [‘*‘I]-A11binding to hypothalamic or brainstem synaptosomes showed that by 60 min binding was at equilibrium in each case. Following each incubation, 1 ml of 4°C Tris HCl buffer (150 mM NaCI, 50 mM Tris, 5 mM EDTA, pH 7.2) was added to each sample which was then centrifuged at 13,000 X 6 for 5 min. The supernatant was removed and the wash repeated. The tip of the microfuge tube containing the pellet was cut and placed in a tube for determination of the radioactivity using a Beckman 5500 gamma counter. Specific binding of [‘251]-AII to synaptosomal membranes was calculated as the mean of triplicate samples obtained by subtracting radioactivity bound in the nonspecific binding tubes from the total binding tubes. and was expressed as fmol/mg protein. The protein content present in the binding assay was determined by the method of Lowry et al. (13). In each individual experiment, brain tissue was obtained from one ( 140-day-old) or two (one-day-old) rats. In each saturation ex~~ment, brain tissue was ob~ined from three ( 14% day-old) or six (one-day-old) rats. Stutisticui and Kinetic Anal~:sc:t
The synaptosome suspensions were treated with one or a combination of the following agents: NE, prazosin. yohimbine, TPA, H-7. Prazosin and yohimbine were used at concentrations of 10 pM and synaptosomes were treated with these agents for 5 min prior to control solution (KRB) or NE. Both prazosin and yohimbine were dissolved in water at initial concentrations of I mM and were then diluted in KRB to the desired concentration. NE was used at concentrations of I-500 PM and synaptosomes were treated with KRB or NE for 60 min prior to analysis of [12SI]-AIIspecific binding. NE was dissolved in KRB at an initial concentration of 10 mM and was diluted in icecold KRB just prior to use. H-7 was used at a concentration of 100 PM and synaptosomes were treated with this agent for 5 min prior to NE or TPA. A stock solution of H-7 was prepared in KRB at 1 mM and was diluted in KRB. TPA was used at a concentration of 0.8 PM, and synaptosomes were treated with this phorbol ester for 60 min prior to analysis of [ ‘2JI]-AII specific binding. Stock solutions of TPA were in DMSO at a concentration of 1 mM and dilutions were made first into water and then into KRB just prior to use. The amounts of DMSO present in the final TPA dilution do not alter [‘251]-AII binding to hypothalamic or brainstem synaptosomes (18). At the end of each incubation. a three-fold volume of ice-coid Krebs-Ringers buffer was added to the treated suspensions, and the synaptosomes were pelleted by centrifugation at 2000 X g for 10 min. The supernatant was aspirated off, the pellet suspended, and the procedure repeated. The fmal pellet was suspended in the sucroseHEPES buffer at a protein concentration of 2-4 mgtml in preparation for AI1 binding. [12Jfl-AII Binding Assay The specific binding of AI1 to synaptosomes was anaiyzed as detailed previously (18). Briefly, 100 ~1 of each synaptosomal suspension containing 200-400 pg protein were incubated with
All data are presented as means I SE. Statistical differences between means were determined using analysis of variance (ANOVA) 1 followed by a Newman-Keuls test where statistical significance was set at p < 0.05 and q values below the 5% level. Kinetic analyses were performed using the EBDA-LIGAND program (Elsevier-Biosoft. Cambridge, U.K.).
-_,J--&--_~
c-i
6
5
4
[NE] -log
“+
3 M
5
4
[NE] -log
3 M
FIG. I. Effects of NE on [‘ZSI]-AIIspecific binding to synaptosomes prepared from the hypothalamus and brainstem. (A) Synaptosornesfmm the hypothalamus (0) or brainstem (0) of one-day-oldrats wereincubated with the indicated ~n~n~~ons of NE for 60 rnin at room tempentture, washed, and [’ Z51]-AIIbinding assays were performed. Values are mean -CSE, with control data plotted on the y axis. * = significantly di&ent from controls. n = 6 experiments, I2 rats total. (B) Synaptosomes from the hvn&alamus(@) or brainstem (0) of f4Odaydi rats were incubated with ihe indicated con~nt~tio~ of NE for 60 min at room temperature, washed, and [‘251]-AIIbinding assays were performed. Values are mean f SE, with control data plotted on the y axis. * = significantly different
from controls. n = 4 experiments, 4 rats total.
ANGIOTENSIN
II BINDING
413
IN SYNAPTOSOMES
TABLE MEAN B,,, AND K., VALUES FOR [‘*‘I]-AI1
1
SPECIFIC BINDING
TO RAT BRAIN SYNAPTOSOMES
Control
H Y PO/one-day-old BST/one-day-old HY PO/ I40-day-old BST/ l40-day-old
NE-Treated Bmar
&mar
ib
ffmol/mg protein)
(nM)
68.2 52.3 34.1 30.2
0.91 I .04 0.95 I.10
t 1.4 ?C9.6 * 3.9 F I.9
k I + L
0.1 I 0. I5 0.25 0.17
(fmol/mg protein) 116.9 + 99.6 i 77.1 I? 51.4 r
0.99 0.89 0.x2 I .01
10.7* Il.9* 6.9; 3.6*
+- 0.25 f 0.23 + 0.13 -t 0.7 I
Synaptosomes prepared from the hypothalamus (HYPO) or brainstem (BST) ofone-day-old or 140-day-old rats were incubated with control solution or NE (50 MM) for 60 min at room temperature. Following this, synaptosomes were washed and [‘ZSI]-AIIbinding saturation assays (0.05-3.0 nM [“SI]-AIl) were performed. Kd and B,,, \alucs Mere calculated bv Scatchard analvsis of saturation data. Values are mean t SE. n = 3 experiments in each case. * Significantly different from controls.
RESUl.TS
Incubation of’ either hypothalamic or brainstem synaptosomes from one-day-old rats with NE (lo-500 PM, 60 min) caused a concentration-dependent increase in [‘2SI]-AII specific binding. In synaptosomes from both areas, a maximal stimulation by NE was obtained at a concentration of 50 PM (Fig. 1A). Below IO PM. NE was ineffective in increasing [‘2SI]-AII specific binding (Fig. IA). Similar results were obtained using synaptosomes prepared from the hypothalamus or brainstem of l40-day-old rats (Fig. I B). Saturation experiments revealed that at all concentrations of [“51]-AII used. NE-treated (50 FM, 60 min) synaptosomes exhibited significantly more binding than control synaptosomes. Scatchard analyses ( 19) (of the saturation data) were linear in both hypothalamic or brainstem synaptosomes prepared from one-day-old rats. In addition, the B,,, values for [“SI]-AII binding in both regions were increased by NE treatment, but the I& values were unaltered (Table I). Similar results were found for synaptosomes prepared from 14Odayold rats (Table 1).
somes. Preincubation of hypothalamic or brainstem synaptosomes from one-day-old rats with H-7 ( 100 FM, 5 min) completely inhibited the increases in [““I]-AI1 binding stimulated by NE (50 PM. 60 min) or by TPA (0.X PM. 60 min) (Table 3). Incubation of synaptosomes with H-7 alone resulted in no changes in [“‘I]-AI1 specific binding. Similar results were obtained for synaptosomes from I4@day-old rats (Table 3). DISCUSSION
The results obtained in the present study lead to the novel observation that NE regulates AI1 binding sites in synaptosomcs prepared from either rat hypothalamus or brainstem. This effect of NE appears to be a stimulation of the number of [“‘I]-AII binding sites rather than their affinity, and the results suggest that NE is an important regulator of All receptors in the brain. This action of NE is mediated by cu,-adrenergic receptors and probably involves activation of PKC subsequent to a stimulation of inositol phospholipid (IP) hydrolysis. Support for this idea
TABLE ,! EFFECTS OF wADRENERGIC RECEPTOR ANTAGONISTS ON NE-STIMULATED INCREASES IN [“‘I]-AI1 SPECIFIC BINDIN(i IN RAT BRAIN SYNAPTOSOMES
Preincubation of hypothalamic or brainstem synaptosomes (from either 1-day-old or 14Oday-old rats) with the ~2,-adrenergic receptor antagomst prazosin ( IO r/M, 5 min) prior to incubation with NE (50 PM, 60 min) completely inhibited the stimulation of [‘*‘I]-AI1 binding elicited by NE (Table 2). Similar preincubations with the cuz-adrenergic receptor antagonist yohimbine were without effect (Table 2). Incubation ofeither hypothalamic or brainstem synaptosomes with prazosin or yohimbine alone had no significant effects on [“SI]-AII specific binding (Table 2).
[‘251]-AII Bound ~fmt~l/mgprotan) One-Day-Old Rats HYPO
BST
19.1 ri- 1.7 15.6 t ?.I 37.8 t 3.4* 31.4 rfr2.0* 21.2 or 2.7 14.9 t I.1 ‘0.1 + 2.9 13.2 * 1.7 35.3 + 2.7* 32.0 i- 3.1* 19.6 -f 2.0 13.9 t 1.6
IJO-Da)-Old Rats HYPO
5.0 f 0.2 12.1 i 1.3* 5.3 2 0.4 4.7 * 0.4 10.3 k I.X* 5.0 zk0.6
Bs7
3.7 8.6 3.9 3.9 8.1 3.2
-+ (1.7 2 0.9’ _L1.4 -t 0.5 + l.O* -I (I.9
in [“51]-.311 Binding in Rut Bruin S~xupto.wme.r
Control NE (50 PM) NE/Prar (10 PM) Pram (10 PM) NE/Yoh (IO/JM) Yoh (IO FM)
In earlier studies, we determined that the phorbol ester TPA increases [‘ZSI]-AII binding in hypothalamic or brainstem synaptosomes ( 18). Furthermore, we showed in neuronal cultures ofhypothalamus and brainstem that either NE or TPA increases [ ‘251]-AII binding, effects mediated via action of protein kinase C ( 1 I, 17). Thus. in this series of experiments, we determined the effects of H-7, a protein kinase C antagonist, on the increase in [‘251]-AIl binding elicited by NE or TPA in brain synapto-
Synaptosomes prepared from the hypothalamus (HY PO) or brainstem (BST) of one-day-old or 140-day-old rats were incubated with control solution or NE in the absence or presence of prazosin (Praz) or yohimbine (Yoh) preincubation for 60 min at room temperature. Following this. synaptosomes were washed and [‘2SI]-AIIbinding assays (0.2 nM [“51]AII) were performed. Values are mean 1. SE. n = 4 experiments (8 total one-day-old rats and 4 total I40-day-old rats). * Significantly different from controls.
l$kct ofFI- 7 on NE- and Phorbol Ester-Stimuhted
incrruses
414
SI?.MNkRS
TABLE 3 EFFECT OF H-7 ON NE- OR TPA-STIMULATED INCREASES IN [“‘I]-AII BINDING IN RAT BRAIN SYNAPTOSOMES I’ZSI1-AIIBoundIfmoVmemoteini One-Day-Old Rats
Control NE (50 fiM) NE/H-7 (100 &I) TPA (0.8 pM) TPA/H-7 (100 rM) H-7 ( 100 PM)
140-Day-Old Rats
HYPO
BST
20.3 f 2.4 39.4 f 2.9*
14.7 I 1.2
5.1 + 0.3
3.5 rt 0.3
28.3 k 4.6*
10.7 + 0.9*
7.7 It OS
5.7 i 0.8
12.2 + 1.3*
3.9 _c 0.3 to.1 i t.t*
4.9 t 0.6 4.8 * 1.2
3.8 7t 1.2 4.3 I 1.4
21.7 f 1.9 15.6 -t 3.1 47.8 f 3.8* 40.3 rt 4.8*
23.1 + 3.7 19.2 f 3.2
15.3 + 2.7 15.0 rt 3.0
HYPO
BST
Synaptosomespreparedfrom the hypothalamus (HYPO) or brainstem (BST) of one-day-old or 140day-old rats were incubated with control solution (KRB),,. NE. or TPA in the absence or oresence of H-7 oreincubation for 60 min at room temperature. Following this, synaptosomes were washed and ~‘zsI]-AIlbinding assays (0.2 nM [‘251]-AIf) were performed. Values are mean f SE. n = 4 ex~~ments (8 total one-day-old rats and 4 total i~day-old rats). * Significantly different from controls.
comes from the fact that the NE-induced increase in [‘2SI]-AII binding is mimicked by the PKC agonist TPA and that the effects of NE and TPA are antagonized by the PKC antagonist H-7. However, we should be cautious in su~e~ng a role for PKC in this NE-induced event because of the relative nonselectivity of H-7 (9, IO), and future studies will utilize the newiy available specific PKC antagonist calphostin C. The involvement of IP hydrolysis in this NE-induced event was suggested because NE concentrations of > 10 PM were required to cause an increase in [‘251]-AII binding, because we determined in other studies that similar concentrations of NE were required to produce a
significant stimulation of IP hydrolysis in the rat brain (4). Our current data shows that there is no qualitative difference in the action of NE in synaptosomes prepared from 1-day-old or adult hypothalamus or brainstem. Thus, the differences in basal lcvcts of AI1 binding that exist in the neonate and adult rat brain (1.3.18) are probably not due to alterations in regulation by NE or PKC at each age. The current results from synaptosomes mirror and confirm our previous findings on the action of NE on AI1 specific binding sites in neuronal cultures of neonate hypothalamus and brainstem (17). The effects of NE on AI1 specific binding in hypothalamus or brainstem synaptosomes or neuronal cultures (17) are rapid, occuring within 60 min. Additionally, in a previous study. we determined that TPA elicits a PKC-de~ndent increase in AI1 receptors in neuronal cultures via a protein synthesis independent mechanism (25). Considering the rapidity of NE’s effects on AH specific binding in hypothalamus or brainstem synaptosomes, and the probable role of PKC, it is likely that protein synthesis is not involved in this NE action. The hypothalamus and brainstem were chosen for review because they contain the highest concentrations of brain AIi receptors (6,8,15,22) and are rich in NE (12.16). Also, there is evidence that in the hypothaIamus NE neurons innervate areas that contain high levels of AI1 receptors (27). Because of the heterogeneity of our crude synaptosomai preparations [i.e., primarily presynaptic but also some postsynaptic elements (7,2 l)]. we cannot specify the exact location of the a,-adrenergic and AI1 receptors and at this point could only speculate. This information is only obtained from studies that determine the exact co-localization of AI1 and cu,-adrenergic receptors in the brain. In conclusion, my studies suggest a role for brain NE in the modulation of central AI1 receptor binding and that cur-adrenergic receptors and protein kinase C are involved in this action. The particular subtype (or subtypes) of brain AII receptor that is altered by NE will be the subject of a future investigation. ACKNOWLEDGMENTS I thank Ida Verzosa for technical assistance and Pia Jacobs for typing the manuscript. This work was supported by NIH grant NS 19441.
REFERENCES 1. Baxter, C.; Horvath, J.; Duggin, G.; Tiller, D. Effect of age on angiotensin II receptors from rat brain. Clin. Sci. 57: 1115-I 135; 1979. 2. Chan, J. Y. H.; Pan, S.; Chan, S. II. H. Participation of noradrenergic neurotransmission in angiotensin H-induced dipsogenic behavior in the rat. Life Sci. 48: 1293- 130 I ; 1991. 3. Cole, F. E.; Blakesley, H. L.; Graci, K. A.; Frohlich, E. D.; MacPhee, A. A. Brain angiotensin II receptor affinity and capacity in SHR and WKY rats: effects of acute dietary changes in NaCl. Brain Res. 190: 272-277; 1980. 4. Crews, F. T.; Raulli, R.; Gonzales, R. A.; Sumners, C.; Raizada, M. K. a,-admnetgic-stimulated responses. J. Cardiovasc. Pharmacol. 11(Suppl. l):S99-S106; 1988. 5. Epstein, A. N.; Fitzsimons, J. T.; Rolls, B. J. Drinking induced by injection of angiotensin into the brain of the rat. J. Physiol. (Lond.) 210~457-474; 1970. 6. Gehlert, D.; Speth, R. C.; Walmsley, J. K. Distribution of [rzsI]angiotensin II binding sites in the rat brain: a quantitative autoradiographic study. Neuroscience 18~837-856; 1986. 7. Gonates, N. K.; Autili~am~tti, L.; Gambetti, P.; Shafer, B. Morphological and biochemical changes in rat synaptosome haetions during neonatal devefopment. J. Cell Biol. 5 1:484-498; 197 1. 8. He&y, D. P.; Maciejewski, A. R.; Printz, M. P. LocaUtion of central angiotensin II receptors with [‘2SI]-Sar’Iles-angiote.nsin II: periventricular sites of the anterior third ventricle. Neuroendoerinology 44: 15-21; 1986.
9. Hidaka, H.; Inagaki, M.; Kawamoto, S.; Sasaki, Y. Isoquinolinesulfonamides: novel and potent inhibitors of protein kinase and protein kinase C. Biochem. 23:5036-5041: 1984. 10. Hidaka, H.; Hagiwara, M. Pharmacology of the isoquinolinesulfonamide protein kinase C inhibitors. Trends Pharmacol. Sci. 8: 162164; 1987. II. Kalberg, C. J.; Sumners, C. Regulation of angiotensin II binding sites in neuronal cultures by protein kinase C. Am. J. Physiol. 258: C61O-c617; 1990. 12. Lindvall, 0.; Bjo~und, A. Dopamine and norep~neph~ne-eontaining neuron systems: their anatomy in the rat brain..In: Emson, P. C., ed. Chemical neuroanatomv. New York Raven Press: 1983: 229-255. 13. Lowry, 0. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin-Phenol reagent. J. BioI. Chem. 193: 265-275; 1951. 14. Mann, J. E E.; SchiBiin, E. L.; Schiller, P. W.; Rascher, W.; Boucher, R.; Genest, J. Cent& actions and brain receptor binding of angiotensin II: influence of sodium intake. Huron 2437-443; 1980. 15. Mendelso~n, F. A. 0.; Quirion, R.; Saavedra, J. M.; Aguilera, G.; Catt, K. J. Autoradiographic localiition of angiotenin II receptors in rat brain, Proc. Natl. Acad. Sci. USA 81: 1575-1579: 1984. 16. Moore, R. Y.; Bloom, F. E. Central catecholamine neuron systems: anatomy and physiology of the norepinephrine and epinephrine systems. Ann. Rev. Neurosci. 2:113-168; 1979.
ANGIOTENSIN
II BINDING
IN SYNAPTOSOMES
17. Myers. L. M.; Sumners. C. Regulation ofangiotensin II binding sites in neuronal cultures by catecholamines. Am. J. Physiol. 257:C706c713: 1989. 18. Myers. L. M.: Raizada, M. K.: Sumners, C. Effects of phorbol esters and a calcium ionophore on angiotensin II binding in rat brain synaptosomes. Neurochem. Res. 14:25-30; 1989. 19. Scatchard. G. The attraction of protein for small molecules and ions. Ann. N.\‘. Acad. Sci. 5 1x%0-672; 1949. 20. Severs. W. B.: Daniels. A. E.: Buckley, J. P. On the central hypertensive elfect of angiotensin II. Int. J. Pharmacol. 6:199-205; 1967. 31. Sgaragli, G. P.; Sen, 1.: Baba. A.: Schulz. R. A.; Cooper. J. R. The mechanism of action of collagcnase on the inhibition of release of acetylcholinc from synaptosomal preparations. Brain Res. 134:I 1% 13; 1977. 22. Sirett. N. E.: Thornton. S. N.: Hubbard, J. 1. Angiotensin binding and pressor activity in the rat ventricular system and midbrain. Brain Res. 166:13Y-148: 1079. 23. Sumner% C.: Phillips M. I. Central injection of angiotensin II alters catecholamine activity in rat brain. Am. J. Physiol. 244:R257-R263; 19x3.
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24. Sumners. C.; Fregly, M. J. Receptors for angiotensin 11.In: Kalimi, M. Y.: Hubbard, J. R.. eds. Peptide hormone receptors, Berlin: W. de Gruyter: 1987664-7 13. 25. Sumners. C.: Rueth, S. M.: Crews. F. ‘T.: Raizada. M. K. Protein kinase C agonists increase the expression of angiotensin II receptors in neuronal cultures. J. Neurochem. 48: l954- I96 I; 1987. 26. Sumners. C.; Myers. L. M.: Kalberg, C. J.: Raizada. M. K. Physiological and pharmacological comparisons of angiotensin II receptors in neuronal and astrocyte glial cultures. Prog. Neurobiol. 34:355385: 1990. 27. Swanson. L. W.: Sawchenko, P. E.: Berod. A.: Hartman. B. K.: Helle. K. B.: Vanorden, D. E. An immunohistochemical study of the organization of catecholaminergic cells and terminaf fields in the pa~~entricular and supraoptic nuclei of the hypothalamus. J. Comp. Neural. 196271~185: 1981. 28. Thomas. W. G.; Sernia. G. Regulation of rat brain angiotensin II (AU) receptors by intravenous All and low8dietary Nat. Brain Res. 345:54-61: 1985.
036 i-9230192 $5.00 t .OO
Vol. 28. pp. 4 I l-4 15. I992
Copyrightc. 1992Pergamon PressLtd.
Norepinephrine Increases Angiotensin II Binding in Rat Brain Synaptosomes COLIN
SUMNERS
Received 15 July 1991 C. ~~?~~r~pi~~p~lr~~~t~ jn~reus~s ~n~l{~I~n~inII ~i~zdin~ in VQIhrcrin .s~~~~~~fu~~(~~t~.s. BRAIN RES BIJLL 28(3) 4 1l-4 15. 199X--Synaptosomes prepared from rat hypothalamus or brainstem contain specific binding sites for [?I-angiotensin II (All). Treatment of these synaptosomes with norepinephrine (NE) (lo-500 PM) for 60 min results in a concentration-dependent increase in [‘2SI]-AII specific binding which appears to be an increase in the number rather than the affinity of these binding sites. This etfect of NE is qualitatively similar in synaptosomes prepared from neonate (one-day-old) or adult (l40-day-old) rats. Furthermore. it is antagonized by prazosin (IO PM) but not by yohimbine (IO JIM), indicating the involvement of a,-adrenergic receptors. Finally, this effect of NE may involve activation of protein kinase C (PKC) because it is mimicked by a PKC agonist (TPA. 0.8 pM; 60 min) and is blocked by a PKC antagonist (H-7. 100 FM). These results match our previous findings on the regulation of AI1 receptors in neurons cultured from the hy~thalamus and brainstem of neonate rats and provide strong evidence for a role of this catecholamine in the modulation of brain AI1 receptors. SUMNERS,
Catecholamines
Angiotensin II receptors
Hypothalamus
Brain stem
Synaptosomes
METHOD
THE brain contains specific receptors for angiotensin II (AII). with highest densities present in the hypothalamus and brainstem (6,8,15,22). AI1 hinds at these receptors and activates neural pathways, including norepinephrine (NE) neurons, ultimately leading to increases in blood pressure and fluid intake (2,5,20.23). The regulation of brain AI1 receptors represents an important way in which the central actions of AI1 are modulated. However, this regulation is di~cult to assess in the in vivo situation due to a variety of different influences that are present, including sodium ions, mineralocorticoids, estrogens, and NE (14.24.26.28). In earlier studies we utilized in vitro primary neuronal cultures prepared from neonate (one-day-old) rat brains as a model system in which to study the regulation of neuronal AI1 receptors [see (26) for review]. We determined that there are numerous regulatory influences on neuronal AI1 receptors, including NE. For example, short-term (0.5-l .O hr) incubation of hypothalamus/hrainstem neuronal co-cultures with NE results in an increased number of [‘251]-AII specific binding sites in these cells (17). This eff‘ect of NE is mediated via ol,-adrenergic receptors and involves activation of inositol phospholipid hydrolysis and protein kinase C (PKC) hut not protein synthesis. In the present study, we determined the effects of NE on the regulation of [12SI]-AII specific binding in another in vitro system, namely synaptosomes prepared from either rat hypothalamus or brainstem. In this preparation. NE elicits increases in [“‘I]AI1 specific binding that are mediated via a,-adrenergic receptors and possibly activation of PKC with no qualitative differences between synaptosomes from neonatal and adult tissue. This data suggests a role for NE in the regulation of brain AI1 receptors.
The animals used in all experiments were male SpragueDawley rats (250-300 g), purchased from Charles River Farms, Wilmington, MA. Two rats were housed per stainless steel cage and allowed free access to water and Purina Laboratory Chow (#SOOl) in a room where both temperature (25°C) and light cycle (07.00 h to 2 I .OO h) were controlled.
Bovine serum albumin fraction V (BSA). N-2-hydroxyethylpiperazine-~-2-ethanesulfonic acid (HEPES), tris (hydroxymethyl) aminomethane (Tris), ethylenediaminetetra-acetic acid (EDTA), angiotensin II (AH), phorbol- I 2-myristate- 13 acetate (TPA). H-7 [ I-(5-isoquinolinylsulfonyl) 2-methyl-piperazine], norepinephrine HCI, prazosin HCI, yohimhine HCl, and dimethylsulfoxide (DMSO) were purchased from Sigma Chemical Co., St. Louis, MO. [‘251]-AII (specific activity 1800 @.Zi/cLg)was purchased from DuPont New England Nuclear, Boston, MA. All other chemicals were purchased from Fisher Scientific, Pittsburgh. PA.
One-day-old or 140-day-old rats were sacrificed by decapitation, and their brains were dissected free and rinsed in icecold 0.9% saline. All studies were conducted between IO:30 and 1 I:30 a.m. For each brain, the brainstem (medulla ohlongata
Requests for reprints should be addressed to Dr. C. Sumners, Department of Physiology. P.O. Box 100274. JHMHC. University of Florida, Gainesville. FI. 376 IO.
411
and pans) and a hypothalamic block containing the supraoptic, paraventricular, anterior, dorsomedial, ventromedial lateral, and posterior nuclei were dissected free from the rest of the brain and all blood vessels and pia mater removed. These areas were used for two reasons: a) They contained the highest levels of brain AI1 receptors and b) these are the areas used in our neuronal cultures, so we were able to make direct comparison with these studies. The Pz pellets, enriched in synaptosomes, were prepared from these two areas basically as described previously (18) according to the method of Sgaragli et al. (21). The brain tissues were immediately homogenized using a teflon homogenizer with five to eight strokes by hand in a cold sucrose-HEPES (0.32 M and 2 mM) buffer, pH 7.6. The homogenates were centrifuged at 1000 X R for 10 min followed by a centrifugation of the supernatants at 12,000 R for 30 min. The pellet resulting from this centrifugation is termed the PZ fraction and represents a crude svnaptosomal fraction. To determine the ]‘Z51]-AII specific binding of this fraction from one-day-old and 140-day-old rats, the hypothalamic block and brainstem pellets were suspended in the sucrose-HEPES buffer at a protein concentration of 2-4 mg/ml, and AI1 binding was performed as detailed below. For the various treatments of the synaptosomal fraction, the pellets were suspended in a small volume of sucrose-HEPES buffer (onetenth the final volume) and then suspended to a final protein concentration of 2-4 mg/ml with a Krebs-Ringer buffer (KRB), which was bubbled with oxygen prior to use ( 139 mM NaCl, 5 mM KCl, 1 mM NaH2P0,, 11 mM NaHCO, . 10 mM dextrose, and 1 mM CaCl,. pH 7.6).
200 ~1 of binding buffer (150 mM NaCl; 50 mM Tris; S mM EDTA, pH 7.2; 0.04% heat-inactivated BSA; and 0.2 nM [ “51JAII) in a m~crofuge tube with (nonsp~ific binding buffer) OF without (total binding buffer) 1 FM unlabeled AI1 for 60 min at room temperature. For saturation experiments, 0.05-3 nM [“‘II-AI1 was used in the incubation mixture. Triplicate samples were used to determine the total and nonspecific binding for each sample. The protein concentration range used during the binding assay fell on the linear region of the binding curve. Time courses of [‘*‘I]-A11binding to hypothalamic or brainstem synaptosomes showed that by 60 min binding was at equilibrium in each case. Following each incubation, 1 ml of 4°C Tris HCl buffer (150 mM NaCI, 50 mM Tris, 5 mM EDTA, pH 7.2) was added to each sample which was then centrifuged at 13,000 X 6 for 5 min. The supernatant was removed and the wash repeated. The tip of the microfuge tube containing the pellet was cut and placed in a tube for determination of the radioactivity using a Beckman 5500 gamma counter. Specific binding of [‘251]-AII to synaptosomal membranes was calculated as the mean of triplicate samples obtained by subtracting radioactivity bound in the nonspecific binding tubes from the total binding tubes. and was expressed as fmol/mg protein. The protein content present in the binding assay was determined by the method of Lowry et al. (13). In each individual experiment, brain tissue was obtained from one ( 140-day-old) or two (one-day-old) rats. In each saturation ex~~ment, brain tissue was ob~ined from three ( 14% day-old) or six (one-day-old) rats. Stutisticui and Kinetic Anal~:sc:t
The synaptosome suspensions were treated with one or a combination of the following agents: NE, prazosin. yohimbine, TPA, H-7. Prazosin and yohimbine were used at concentrations of 10 pM and synaptosomes were treated with these agents for 5 min prior to control solution (KRB) or NE. Both prazosin and yohimbine were dissolved in water at initial concentrations of I mM and were then diluted in KRB to the desired concentration. NE was used at concentrations of I-500 PM and synaptosomes were treated with KRB or NE for 60 min prior to analysis of [12SI]-AIIspecific binding. NE was dissolved in KRB at an initial concentration of 10 mM and was diluted in icecold KRB just prior to use. H-7 was used at a concentration of 100 PM and synaptosomes were treated with this agent for 5 min prior to NE or TPA. A stock solution of H-7 was prepared in KRB at 1 mM and was diluted in KRB. TPA was used at a concentration of 0.8 PM, and synaptosomes were treated with this phorbol ester for 60 min prior to analysis of [ ‘2JI]-AII specific binding. Stock solutions of TPA were in DMSO at a concentration of 1 mM and dilutions were made first into water and then into KRB just prior to use. The amounts of DMSO present in the final TPA dilution do not alter [‘251]-AII binding to hypothalamic or brainstem synaptosomes (18). At the end of each incubation. a three-fold volume of ice-coid Krebs-Ringers buffer was added to the treated suspensions, and the synaptosomes were pelleted by centrifugation at 2000 X g for 10 min. The supernatant was aspirated off, the pellet suspended, and the procedure repeated. The fmal pellet was suspended in the sucroseHEPES buffer at a protein concentration of 2-4 mgtml in preparation for AI1 binding. [12Jfl-AII Binding Assay The specific binding of AI1 to synaptosomes was anaiyzed as detailed previously (18). Briefly, 100 ~1 of each synaptosomal suspension containing 200-400 pg protein were incubated with
All data are presented as means I SE. Statistical differences between means were determined using analysis of variance (ANOVA) 1 followed by a Newman-Keuls test where statistical significance was set at p < 0.05 and q values below the 5% level. Kinetic analyses were performed using the EBDA-LIGAND program (Elsevier-Biosoft. Cambridge, U.K.).
-_,J--&--_~
c-i
6
5
4
[NE] -log
“+
3 M
5
4
[NE] -log
3 M
FIG. I. Effects of NE on [‘ZSI]-AIIspecific binding to synaptosomes prepared from the hypothalamus and brainstem. (A) Synaptosornesfmm the hypothalamus (0) or brainstem (0) of one-day-oldrats wereincubated with the indicated ~n~n~~ons of NE for 60 rnin at room tempentture, washed, and [’ Z51]-AIIbinding assays were performed. Values are mean -CSE, with control data plotted on the y axis. * = significantly di&ent from controls. n = 6 experiments, I2 rats total. (B) Synaptosomes from the hvn&alamus(@) or brainstem (0) of f4Odaydi rats were incubated with ihe indicated con~nt~tio~ of NE for 60 min at room temperature, washed, and [‘251]-AIIbinding assays were performed. Values are mean f SE, with control data plotted on the y axis. * = significantly different
from controls. n = 4 experiments, 4 rats total.
ANGIOTENSIN
II BINDING
413
IN SYNAPTOSOMES
TABLE MEAN B,,, AND K., VALUES FOR [‘*‘I]-AI1
1
SPECIFIC BINDING
TO RAT BRAIN SYNAPTOSOMES
Control
H Y PO/one-day-old BST/one-day-old HY PO/ I40-day-old BST/ l40-day-old
NE-Treated Bmar
&mar
ib
ffmol/mg protein)
(nM)
68.2 52.3 34.1 30.2
0.91 I .04 0.95 I.10
t 1.4 ?C9.6 * 3.9 F I.9
k I + L
0.1 I 0. I5 0.25 0.17
(fmol/mg protein) 116.9 + 99.6 i 77.1 I? 51.4 r
0.99 0.89 0.x2 I .01
10.7* Il.9* 6.9; 3.6*
+- 0.25 f 0.23 + 0.13 -t 0.7 I
Synaptosomes prepared from the hypothalamus (HYPO) or brainstem (BST) ofone-day-old or 140-day-old rats were incubated with control solution or NE (50 MM) for 60 min at room temperature. Following this, synaptosomes were washed and [‘ZSI]-AIIbinding saturation assays (0.05-3.0 nM [“SI]-AIl) were performed. Kd and B,,, \alucs Mere calculated bv Scatchard analvsis of saturation data. Values are mean t SE. n = 3 experiments in each case. * Significantly different from controls.
RESUl.TS
Incubation of’ either hypothalamic or brainstem synaptosomes from one-day-old rats with NE (lo-500 PM, 60 min) caused a concentration-dependent increase in [‘2SI]-AII specific binding. In synaptosomes from both areas, a maximal stimulation by NE was obtained at a concentration of 50 PM (Fig. 1A). Below IO PM. NE was ineffective in increasing [‘2SI]-AII specific binding (Fig. IA). Similar results were obtained using synaptosomes prepared from the hypothalamus or brainstem of l40-day-old rats (Fig. I B). Saturation experiments revealed that at all concentrations of [“51]-AII used. NE-treated (50 FM, 60 min) synaptosomes exhibited significantly more binding than control synaptosomes. Scatchard analyses ( 19) (of the saturation data) were linear in both hypothalamic or brainstem synaptosomes prepared from one-day-old rats. In addition, the B,,, values for [“SI]-AII binding in both regions were increased by NE treatment, but the I& values were unaltered (Table I). Similar results were found for synaptosomes prepared from 14Odayold rats (Table 1).
somes. Preincubation of hypothalamic or brainstem synaptosomes from one-day-old rats with H-7 ( 100 FM, 5 min) completely inhibited the increases in [““I]-AI1 binding stimulated by NE (50 PM. 60 min) or by TPA (0.X PM. 60 min) (Table 3). Incubation of synaptosomes with H-7 alone resulted in no changes in [“‘I]-AI1 specific binding. Similar results were obtained for synaptosomes from I4@day-old rats (Table 3). DISCUSSION
The results obtained in the present study lead to the novel observation that NE regulates AI1 binding sites in synaptosomcs prepared from either rat hypothalamus or brainstem. This effect of NE appears to be a stimulation of the number of [“‘I]-AII binding sites rather than their affinity, and the results suggest that NE is an important regulator of All receptors in the brain. This action of NE is mediated by cu,-adrenergic receptors and probably involves activation of PKC subsequent to a stimulation of inositol phospholipid (IP) hydrolysis. Support for this idea
TABLE ,! EFFECTS OF wADRENERGIC RECEPTOR ANTAGONISTS ON NE-STIMULATED INCREASES IN [“‘I]-AI1 SPECIFIC BINDIN(i IN RAT BRAIN SYNAPTOSOMES
Preincubation of hypothalamic or brainstem synaptosomes (from either 1-day-old or 14Oday-old rats) with the ~2,-adrenergic receptor antagomst prazosin ( IO r/M, 5 min) prior to incubation with NE (50 PM, 60 min) completely inhibited the stimulation of [‘*‘I]-AI1 binding elicited by NE (Table 2). Similar preincubations with the cuz-adrenergic receptor antagonist yohimbine were without effect (Table 2). Incubation ofeither hypothalamic or brainstem synaptosomes with prazosin or yohimbine alone had no significant effects on [“SI]-AII specific binding (Table 2).
[‘251]-AII Bound ~fmt~l/mgprotan) One-Day-Old Rats HYPO
BST
19.1 ri- 1.7 15.6 t ?.I 37.8 t 3.4* 31.4 rfr2.0* 21.2 or 2.7 14.9 t I.1 ‘0.1 + 2.9 13.2 * 1.7 35.3 + 2.7* 32.0 i- 3.1* 19.6 -f 2.0 13.9 t 1.6
IJO-Da)-Old Rats HYPO
5.0 f 0.2 12.1 i 1.3* 5.3 2 0.4 4.7 * 0.4 10.3 k I.X* 5.0 zk0.6
Bs7
3.7 8.6 3.9 3.9 8.1 3.2
-+ (1.7 2 0.9’ _L1.4 -t 0.5 + l.O* -I (I.9
in [“51]-.311 Binding in Rut Bruin S~xupto.wme.r
Control NE (50 PM) NE/Prar (10 PM) Pram (10 PM) NE/Yoh (IO/JM) Yoh (IO FM)
In earlier studies, we determined that the phorbol ester TPA increases [‘ZSI]-AII binding in hypothalamic or brainstem synaptosomes ( 18). Furthermore, we showed in neuronal cultures ofhypothalamus and brainstem that either NE or TPA increases [ ‘251]-AII binding, effects mediated via action of protein kinase C ( 1 I, 17). Thus. in this series of experiments, we determined the effects of H-7, a protein kinase C antagonist, on the increase in [‘251]-AIl binding elicited by NE or TPA in brain synapto-
Synaptosomes prepared from the hypothalamus (HY PO) or brainstem (BST) of one-day-old or 140-day-old rats were incubated with control solution or NE in the absence or presence of prazosin (Praz) or yohimbine (Yoh) preincubation for 60 min at room temperature. Following this. synaptosomes were washed and [‘2SI]-AIIbinding assays (0.2 nM [“51]AII) were performed. Values are mean 1. SE. n = 4 experiments (8 total one-day-old rats and 4 total I40-day-old rats). * Significantly different from controls.
l$kct ofFI- 7 on NE- and Phorbol Ester-Stimuhted
incrruses
414
SI?.MNkRS
TABLE 3 EFFECT OF H-7 ON NE- OR TPA-STIMULATED INCREASES IN [“‘I]-AII BINDING IN RAT BRAIN SYNAPTOSOMES I’ZSI1-AIIBoundIfmoVmemoteini One-Day-Old Rats
Control NE (50 fiM) NE/H-7 (100 &I) TPA (0.8 pM) TPA/H-7 (100 rM) H-7 ( 100 PM)
140-Day-Old Rats
HYPO
BST
20.3 f 2.4 39.4 f 2.9*
14.7 I 1.2
5.1 + 0.3
3.5 rt 0.3
28.3 k 4.6*
10.7 + 0.9*
7.7 It OS
5.7 i 0.8
12.2 + 1.3*
3.9 _c 0.3 to.1 i t.t*
4.9 t 0.6 4.8 * 1.2
3.8 7t 1.2 4.3 I 1.4
21.7 f 1.9 15.6 -t 3.1 47.8 f 3.8* 40.3 rt 4.8*
23.1 + 3.7 19.2 f 3.2
15.3 + 2.7 15.0 rt 3.0
HYPO
BST
Synaptosomespreparedfrom the hypothalamus (HYPO) or brainstem (BST) of one-day-old or 140day-old rats were incubated with control solution (KRB),,. NE. or TPA in the absence or oresence of H-7 oreincubation for 60 min at room temperature. Following this, synaptosomes were washed and ~‘zsI]-AIlbinding assays (0.2 nM [‘251]-AIf) were performed. Values are mean f SE. n = 4 ex~~ments (8 total one-day-old rats and 4 total i~day-old rats). * Significantly different from controls.
comes from the fact that the NE-induced increase in [‘2SI]-AII binding is mimicked by the PKC agonist TPA and that the effects of NE and TPA are antagonized by the PKC antagonist H-7. However, we should be cautious in su~e~ng a role for PKC in this NE-induced event because of the relative nonselectivity of H-7 (9, IO), and future studies will utilize the newiy available specific PKC antagonist calphostin C. The involvement of IP hydrolysis in this NE-induced event was suggested because NE concentrations of > 10 PM were required to cause an increase in [‘251]-AII binding, because we determined in other studies that similar concentrations of NE were required to produce a
significant stimulation of IP hydrolysis in the rat brain (4). Our current data shows that there is no qualitative difference in the action of NE in synaptosomes prepared from 1-day-old or adult hypothalamus or brainstem. Thus, the differences in basal lcvcts of AI1 binding that exist in the neonate and adult rat brain (1.3.18) are probably not due to alterations in regulation by NE or PKC at each age. The current results from synaptosomes mirror and confirm our previous findings on the action of NE on AI1 specific binding sites in neuronal cultures of neonate hypothalamus and brainstem (17). The effects of NE on AI1 specific binding in hypothalamus or brainstem synaptosomes or neuronal cultures (17) are rapid, occuring within 60 min. Additionally, in a previous study. we determined that TPA elicits a PKC-de~ndent increase in AI1 receptors in neuronal cultures via a protein synthesis independent mechanism (25). Considering the rapidity of NE’s effects on AH specific binding in hypothalamus or brainstem synaptosomes, and the probable role of PKC, it is likely that protein synthesis is not involved in this NE action. The hypothalamus and brainstem were chosen for review because they contain the highest concentrations of brain AIi receptors (6,8,15,22) and are rich in NE (12.16). Also, there is evidence that in the hypothaIamus NE neurons innervate areas that contain high levels of AI1 receptors (27). Because of the heterogeneity of our crude synaptosomai preparations [i.e., primarily presynaptic but also some postsynaptic elements (7,2 l)]. we cannot specify the exact location of the a,-adrenergic and AI1 receptors and at this point could only speculate. This information is only obtained from studies that determine the exact co-localization of AI1 and cu,-adrenergic receptors in the brain. In conclusion, my studies suggest a role for brain NE in the modulation of central AI1 receptor binding and that cur-adrenergic receptors and protein kinase C are involved in this action. The particular subtype (or subtypes) of brain AII receptor that is altered by NE will be the subject of a future investigation. ACKNOWLEDGMENTS I thank Ida Verzosa for technical assistance and Pia Jacobs for typing the manuscript. This work was supported by NIH grant NS 19441.
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