Regulation of rat brain angiotensin II (AII) receptors by intravenous AII and low dietary Na+

54

Bratn Research. 345 1i~5) 54- hi

BRE 11056

Regulation of Rat Brain Angiotensin II (All) Receptors by Intravenous AII and Low Dietary Na ÷ WALTER G. THOMAS and CONRAD SERNIA Department of Physiology and Pharmacology, University of Queensland, St. Lucia 4067 (Australia) (Accepted January 3rd, 1985) Key words: angiotensin receptor - - brain - - renin-angiotensinsystem - - receptor regulation

Previous studies have shown the presence of specific All receptors at several areas of the brain. The purpose of this study was to examine by radioreceptor assay the effect of intravenous AII infusion (5 or 25 ng/kg/min) and low dietary Na ÷ (<8 mmoF100 g) on All receptors in five brain regions: the olfactory lobes (OLF), hypothalamusdthalamusdseptum(HTS), midbrain (MID), cerebellum (CER) and medulla (MED). Scatchard analysis of binding data from control rats showed significant (P < 0.01 ANOVA} differences between brain areas in both K~ (1.54 OLF, 1.87 HTS. 1.25 MID. t .33 MED, 0.77 CER x 109 M-0 and R o (32I OLF, 224 FITS. 203 MID, 145 MED. 41 CER fmol/g tissue). Following the i,v. infusion of AII for 4-7 days, marked changes were observed in the areas with a porous BBB. the HTS and MED. Both the K a [3.20 (HTS) and 0.67 (MED) x 109 M-l] and Ro [116 (HTS) and 249 (MED) fmol/g tissue] changed. In addition, decreases in Ro were also observed in the OLF (241 fmol/g tissue) and CER (21 fmol/g tissue), areas which have not been considered as being accessible to blood-borne AII. A low Na + diet for 21-30 days changed the Ka and R o in all five regions but not in similar directions. Furthermore, with the exception of the OLF the direction of change was not similar to that caused by i.v. infusion of AII. It was concluded that AII receptor sites in the rat brain differ from each other in both receptor properties and in their response to such regulatory factors as AII and Na ÷ depletion. In addition, the division of receptor sites into those within and those outside the BBB appears to lack functional significance since both types were affected by t.v. exogenous All

INTRODUCTION

instead be affected by an intrinsic A l l - g e n e r a t i n g mechanism~2.23.

The octapeptide h o r m o n e , angiotensin II ( A l l ) has several k n o w n actions on the brain including in-

The physiological importance or the relevance of these n u m e r o u s discrete sites of A l l action ts presently not well understood. It has. however, been shown that decreasing sodium intake will decrease blood pressure and water intake in both n o r m a l and spontaneously hypertensive rats (SHR) and that the changes correspond to receptor changes in the hypothalamus/thalamus/septum (HTS) area which contains the A l l receptor sites in subfornical organ and organum vasculosum of the lamina terminalis ~0.tS.

creases in water intake, blood pressure and release of vasopressin and ACTH16.18.22.26-27. These actions appear to be mediated by some of the circumventricular areas in the forebrain and medulla where the blood brain barrier (BBB) is permeable to A l l and therefore accessible to blood AII2t,26. Specific A l l receptors have been localized at these sites by autoradiography, immunofluorescence and radioreceptor assay 3.7,17,27.29. However. specific A l l receptors have also been found outside the circumventricular organs at sites in the thalamus, hypothalamus, pituitary, septum, midbrain, cerebellum3,4.6,27, 28 and, as recently observed, the olfactory lobes s,14. These sites are believed to be inaccessible to blood A l l and may

These observations leave little doubt that brain A l l receptors in at least some sites are, like A l l receptors in peripheral tissues, sensitive to changes in the sodium intake. In the present study, these observations on receptor regulation were extended by including both low dietary sodium and low doses of A l l infused

Correspondence: C Sernia. Department of Physiology and Pharmacology, University of Queensland. St. Lucia, 4067 Australia. 00~-8993/85/$03.30 © 1985 Elsevier Science Publishers B .V. (Biomedical Division)

55 intravenously at constant rate by osmotic minipumps. In addition, the effects of these treatments were examined concurrently in five brain regions, including areas accessible to blood-borne All (HTS and medulla) as well as areas thought inaccessible to the hormone (olfactory lobes, midbrain, cerebellum)8,14,27,29.

recorded for all animals.

Preparation of brain membranes

Synthetic [Asp 1,IleS]AII, [Asp I N a p ] AII, AIII, AI and [Sarl-AlaS]AII were obtained from Chemalog (South Plainfield, NJ); Na[125I] from Amersham (Sydney, Australia); bovine serum albumin (BSA), dithiothreitol (DTT), phenylmethylsulfonyl fluoride (PMSF), bacitracin and 2,3-dimercaptopropano! (BAL) were supplied by Sigma (St. Louis, MO). [125I]Monoido-AII was prepared by the chloramine-T method and purified on a column (1.2 x 80 cm) of DEAE-Sephadex A-2519. The specific activity of the [125I]AII was 1200-1400 Ci/g AII.

Rats were killed by ether overdose and the brain removed and placed on ice. Five gross areas were dissected as described previouslyZS; the cerebral cortex, the olfactory bulbs (OLF), the hypothalamus/thalamus/septum (HTS), the midbrain (MID), the medulla (MED), and the cerebellum (CER). Each area was weighed and homogenized in 40 vols. of 50 mM Tris-HC1 buffer (pH 7.4, 4 °C) with a Polytron (Brinkman Instruments, Westburg, NY; 4 x 5 s on setting 5). The particulate fraction was collected by ultracentrifugation at 50,000 g for 20 min, resuspended with more Tris-HCl buffer and recentrifuged. The pellet was then resuspended in assay buffer (10 mM Na2HPOa, 120 mM NaC1, 5 mM EDTA, 0.3 mM PMSF, pH 7.2 at 4 °C) and left for 30 rain at 4 °C. The suspension was centrifuged at 50,000 g for 20 min once more to obtain the membrane pellet to be used in radioreceptor assays. This procedure is a modification of that of Bennett and Snyder 4.

Treatments

Radioreceptor and radioimmunoassays

Adult male Wistar rats (250-350 g) were used for all experiments. They were obtained from the University of Queensland central breeding house and maintained on standard commercial rat food containing 28 mmol Na+/100 g and 14 mmol K+/100 g as measured by flame photometry, and tap water. The first treatment consisted of feeding for 21 days a low Na + diet (8 mmol Na+/100 g; 14 mmol K+/100 g) made in the laboratory according to the formula used by Clements et al. 9. To avoid any possible effect of changing from a commercial to a laboratory-made diet, rats were fed the latter diet containing the same Na + and K + content as the commercial source for 10 days before changing to the Na+-deficient diet. The second treatment consisted of a group of 25 rats injected with 5 or 25 ng AII/kg/min for 4 - 7 days by means of osmotic minipumps (Alzet, Palo Alto, CA) which were implanted subcutaneously and connected by a catheter to the left external jugular vein. A similar number of rats were infused with isotonic saline and.served as paired controls. All surgery was performed under anesthesia induced by the i.p. injection of 50 mg/kg sodium ('Sagatal', May and Baker, Sydney, Australia). The daily water intake was

Freshly prepared brain membranes were suspended in the assay buffer containing 5 mM DTT, 0.1 mM bacitracin and 0.2% BSA, to a dilution of 100-250 mg tissue/ml. For the radioreceptor assay, 100 ktl of this membrane suspension was added to 100 ul of AII (0.05-8 nM) and 50ktl of [125I]AII (2-2.5 x 104 CPM), mixed thoroughly and left for 60 min at room temperature. The suspension was filtered on GF/C discs (Whatman) and washed with 8 ml of phosphate-buffered saline. The bound radioactivity was measured in a y-counter (LKB 1124, 50% efficiency) and the results were analyzed by a Scatchard plot 24 to obtain the affinity (Ka) and receptor capacity (R0). The AII bound at the 8 nM concentration served as a measure of non-specific binding. The co-efficient of variation for this radioreceptor assay was 1.7 + 0.3% (n = 23) and the specificity of binding, compared to [IleS]AII, was 1.2, 0.82, 0.68 and 0.05 for [Val5]AII, [Sar~,AlaS]AII, A I I I and AI, respectively. For all the brain areas except the HTS, tissue from single rats was insufficient to allow a saturation curve to be obtained. Therefore, for these areas tissue from three rats was pooled for each estimation of K a and Ro. The protein content of each area was measured by the

MATERIALS AND METHODS

Reagents

56

Keuls multiple range test. All data were expressed z~s mean + S.E.

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RESULTS

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Fig. I. A: daily water intake in a group of 9 rats for 5 days be[ore and 20 days after being placed on a diet containing < 8 mmol Na+/100 g. B: plasma renin activity after 5 days of AH infusion (25 ng/kg/min) and 20 days of Na ÷ depletion. C: plasma sodium concentration in control and treated rats. (All data are shown as mean + S.E.; *P < 0.05, **P < 0.01, ***P < 0.001.)

Coomassie Blue method 5. The plasma renin activity (PRA) was measured in control and experimental animals. Plasma was separated from blood withdrawn from the inferior vena cava just before death by ether overdose and mixed with E D T A to a concentration of 15 mM. This plasma was diluted 1:2 with 100 mM phosphate containing 15 mM E D T A , 5 mM B A L and 0.3 mM PMSF (pH 6.5) and incubated at 37 °C for 1 h with a paired control at 4 °C. The A I released was measured by a radioimmunoassay described previously25 and the results expressed as ng AI/ml plasma/h.

Statistics Each experiment included both control and treatment rats. Mean values were compared by paired ttest, or, where more than two groups were involved, by an analysis of variance followed by a Newman-

The daily water intake of Na+ replete animals was 26.6 + 2.8 ml/rat/day. When changed to a Na--deficient diet, drinking decreased markedly for the first day but then steadily increased to and remained at approximately 78% of control intake (Fig. 1A). The intravenous infusion of All had no effect on water intake apart from a transient decrease immediately following surgery (not shown). The mean PRA for control rats was 13.6 - t.8 ng AI/ml plasma/h. After 5 days infusion of A l l at 25 ng AII/kg/min it decreased by 34% to 9.0 z 1.2 ng AI/ml/h (P < 0.05. Fig. 1B). The Na+-deficient diet increased PRA by 347% of control to 47.2 _+ 5.9 ng AI/ml/h. The plasma Na+ concentration was t44.3 _ 1.2 mM in control rats. Both the All infusion (25 ng AII/kg/min) and Na ÷ restriction decreased plasma Na + significantly to 138.1 Jr 2.3 and 139.7 ~- 2.1 raM. respectively. At the lower dose of All (5 ng AII/kg! min) no change in plasma Na + was observed Ct45.1 + 1.4 mM).

Specific binding of [125I]AH The binding of [125I]AII to membranes (12.5 mg/ml) from the hypothalamus/thalamus/septum/midbrain (HTSM) increased with time and reached a maximum between 60 and 75 min (Fig. 2A). Beyond this incubation time some decrease in specific binding was evident, presumably due to enzymatic degradation of [125I]AII. Having established the optimal incubation time. a 60-min incubation at room temperature was used to examine the effect of tissue concentration on binding (Fig. 2B). An approximate linear relationship was found between specific binding and tissue concentration up to 20 mg tissue/ml. Based on these results all radioreceptor assays were performed with 10-20 mg tissue/ml. Binding of AII to the olfactory lobe, HTS, midbrain, medulla and cerebellum was analysed by Scatchard plot and resolved into an affinity constant (K,) and receptor concentration (Ro) (Fig. 3). The cerebral cortex was also examined and found to lack specific binding. The results from saline control rats

57

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Fig. 2. Specific binding of [:251]AIIto brain tissue. A: increase in binding with time to homogenate from the HTS (12.5 mg tissue/tube). B: relationship between [125I]AIIbinding and tissue weight. Incubationwas at 20 °C. Each point is the mean of triplicate determinations from two separate experiments.

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show that significant differences in both K a and Ro are present between brain areas (Fig. 3A and B). The K, ranged from the highest of 1.87 + 0.15 x 109 M-1 in the HTS to the low value of 0.77 + 0.08 x 109 M -1 in the cerebellum. A N e w m a n - K e u i s multiple comparison test (inset of Fig. 3B) shows that the cerebellum is significantly different from all other areas and that the affinities of the medulla and midbrain are also different from each other. More marked differences were observed in the R o with an 8-fold range between the olfactory lobe (321 + 55 fmol/g tissue) and the cerebellum (41 + 7 fmol/g tissue). The data have also been expressed as fmol/mg protein because of the consistently higher content measured in the HTS (mean of 45 mg/g tissue, n = 11) compared to the olfactory bulb (34 mg/g tissue) midbrain (27 mg/g tissue), medulla (32 mg/g tissue) and cerebellum (31 mg/g tissue). The higher protein content in the HTS alters the relative distribution of receptors in the five brain regions. A H infusion The binding of A l l to the five brain areas after a

4 - 7 - d a y infusion of 25 ng AII/kg/min was also ana-

Fig. 3. The affinity (K,) and receptor concentration (Ro) of the olfactory bulb (OLF), hypothalamus/thalamus/septum (HTS), midbrain (MID), cerebellum (CER) and medulla (MED). R o has been expressed both as fmol/g tissue (Q) and fmol/mg protein (N). The statistical comparison of brain areas by a Newman-Keuls test is shown as an inset in each figure and the number of experiments per group is shown at the bottom of each column. Each experiment consisted of tissue pooled from 3 rats except for the HTS. Each estimate of K, and Ro required tissue from 3 rats except for the HTS area where sufficient tissue could be obtained from 1 rat. (*P < 0.05. **P < 0.01, ***P < 0.001 .)

lyzed by Scatchard plot and expressed as a percentage of the paired saline control value (Fig. 4). The areas examined did not respond in a similar way to the A l l infusion. The K a changed in the HTS and medulla but not in the other areas. For the HTS an increase to 171 + 19% of control was observed, in contrast to a sharp decrease in the medulla to 53 _+ 4% of control. The K, for the olfactory bulb, midbrain and cerebellum remained unchanged. The A I I infusion affected the Ro of all five areas with the exception of the midbrain (Fig. 4C). In the HTS, cerebellum and olfactory bulb respectively, the R o decreased to 69 + 4%, 50 + 11% and 75% (n = 2; 12 rats) that of con-

5~ trol while in the medulla an increase to 172 __+ 19% of control was observed. The effect of a lower infusion rate of AII (5 ng/kg/min) was investigated in the HTS and cerebellum. The changes were similar to those for the higher dose rate but smaller in magnitude. Besides the four experiments shown in Fig. 4 for the cerebellum, a further 3 experiments were performed (at the higher

K a were found in the midbrain (199 _+ 13%), medulla (150 + 13%) and cerebellum (154 _+ 2 t % ) while a decrease was measured in the HTS (70 _+ 9f,; ). Large changes in Ro were also found, with the midbrain, cerebellum and medulla decreasing to 55 :± 12%, 54 _+ 6% and 64 + 6%, respectively, and the HTS increasing to 149 + 28% of control. The olfactory b u l b was the least affected by both the A l l infusion and

A l l infusion rate) where the binding decreased to be-

Na + depletion, with a decrease in R o to 75% of con-

low the sensitivity of the radioreceptor assay.

trol and no change in K~. While the n u m b e r of experiments for this area was small (a total of 24 rats in 4 ex-

Na + dq~letion

periments) the close agreement between experi-

A low Na ÷ diet fed for 2 1 - 3 0 days affected A l l receptors in all five areas (Fig. 5). Marked increases in

ments suggests that the data are reliable estimates of treatment effect.

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Fig. 4. The effect of 4-7 days i.v. infusion of All (25 ng/kg/min) by osmotic minipumps on brain All receptors. A: representative Scatchard plots from saline-infused (O)and AII,infused rats (©). B and C: the Ka and Ro of All receptors from Allinfused rats expressed as a percentage of the value from paired saline-infused rats. Control and treatment groups were compared by paired t-test (*P < 0.05, **P < 0.01, ***P < 0,00i); the number of estimates is indicated at the bottom of each column. OLF, olfactory lobe; HTS, hypothalamusdthalamus/ septum; MID, midbrain; CER, cerebellum; MED, medulla.

Fig. 5. The effect of 21-30 days low dietary Na + on brain All receptors. A: representative Scatclmrd plots from-saline-infused (0) and All.infused rats (©). B and C. The Ka and Ro of AII receptors from AII-infused rats expressed as a percemage of the value from paired saline-infused rats. Control and treatment groups were compared by paired t-test f*P < 0.05. **P < 0.01, ***P < 0.001); the number of estimates is indicated at the bottom of each column. OLF. olfactory lobe; FITS, hypothalamus/thalamus/septum: MID: midbrain; CUR, cerebellum; MED, medulla.

59 DISCUSSION It is now well established that the rat brain has AII receptors in distinct areas of the forebrain, midbrain, cerebellum and medulla oblongata3, ~,6,8A4,18,27,29. Higher receptor densities have been found in the hypothalamus, thalamus, septum and midbrain areas although the precise order of binding capacity varies between reportsS,lS, 27 and according to the way the results have been expressed (Fig. 3B). Harding et al.14 reported a high specific binding in the olfactory lobe, a finding which has since been confirmed by others 8 as well as by this study where it was found to contain the highest receptor concentration (Fig. 3B). With one exception 2, reports which have examined the topographical distribution of All receptors have measured it as binding 'activity' without being able to distinguish between receptor affinity (Ka) and receptor number (Ro). By pooling tissue from three rats it became possible to analyze each area in terms of K~ and R o and by these means to establish that large differences (8-fold between the cerebellum and olfactory lobe) in Ro were present between brain areas. The values of Ro for the HTS and midbrain area in our study are similar to those reported recently for the combined HTS/midbrain by Cole et al.10 (233 fmol/g tissue). Between area differences in K a were also present in our study although the range of values, from 0.77 × 109 M -1 for the cerebellum to 1.87 x 109 M -1 for the HTS was not particularly large. This limited range in K a probably explains the failure by Baxter et al. 2 to observe differences in K a between the thalamus, hypothalamus, midbrain and medulla from single estimates of each area. The AII receptor is known to be regulated by its homologous h o r m o n e 1,n,13,15. However, the changes induced by AII are not similar for all All receptive tissues but involves increases in Ro in some (e.g. adrenal) and decreases in others (e.g. vascular and uterine smooth muscle) 1,11,15. Compared to these tissues the brain presents a more complex situation because of the localization of receptors into several distinct areas within the one tissue and the presence of the BBB at some (midbrain, cerebellum and olfactory bulb) of these areas. From previous reports on the impermeability of the BBB to AII it was expected that AII infusion at the low doses used would not af-

fect All receptors at sites within the B B B 20,21,27,29. This was not the case; there were significant decreases in Ro for the cerebellum and the olfactory lobe although the midbrain remained unaffected. One of these areas, the cerebellum, was extremely sensitive to AII infusion, showing a decrease in binding to below the level of reliability of our radioreceptor assay in 3 of 7 experiments. The HTS and medulla, both containing areas of porous BBB, responded by changes in both Ko and R o but whereas the K a increased and R o decreased in the HTS, the opposite occurred in the medulla. These changes occurred at infusion rates of AII which should have raised plasma AII by only 2-3-fold1, n. These results establish the peripheral renin-angiotensin system as a potent regulator of brain AII receptors and they reaffirm the view that the elicitation of thirst, centrally mediated increases in blood pressure and stimulation of vasopressin release occur within the physiological range of AII concentration. The cerebellum and olfactory lobe also appear to be within the regulation of bloodborne AII, although little is known of the action of All on these areas8, 28. Low dietary Na + affected the AII receptors in all five areas (Fig. 5, Table I). The K a increased significantly in the cerebellum, midbrain and medulla, and decreased in the HTS. R o decreased in all areas except the medulla and HTS where it decreased. As was the case for All infusion, brain All receptors do not respond uniformily to a regulatory factor but each AII receptor site behaves independently of the others. These changes in receptors should be reflected in the sensitivity to circulating AII and this appears to be so for the responses that have been tested, namely water intake and blood pressure10, ~s.

TABLE I Summary o f changes in K a and R o following i.v. infusion o f A II (25 ng/kg/min) by osmotic minipumps f o r 4 - 7 days, or a low Na ÷ diet for 2 1 - 3 0 days Brain area

Olfactory lobe HTS Midbrain Cerebellum Medulla

A H infusion

Na + depletion

Ka

Ro

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Ro

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60 These two responses to A I I are blunted by Na ÷ depletion and are in accord with the decrease in water intake in our experiments (Fig. 1). However, in our study, R o for the HTS was increased while two other reports found Ro to be decreased s,10. This discrepan-

possible that a transport mechanism like that for insulin exists 19 and it may even be speculated that the "brain' r e n i n - a n g i o t e n s i n system becomes activated 12,23. Alternatively, the observed receptor

cy may be partly explained by the inclusion in other

changes may not be due to a direct effect of A l l and thus the presence of A I I in the immediate vicinity of

studies of the midbrain in the tissue block used

the receptors would not be necessary,

(HTSM) which behaves in the opposite way to the HTS (Fig. 5, Table I).

Whatever be the merits of these possibilities, our results indicate that the functional division of A l l re-

A comparison of the effect of A I I infusion and Na +

ceptor areas into those outside (accessible to peripheral A l l ) and those inside (accessible only to locally

depletion reveals a n u m b e r of i m p o r t a n t features (Table I). Firstly, the effects of the two treatments on a particular brain area are not similar, with the exception of the olfactory bulbs. If the elevation of A l l concentration, whether by injection of exogenous A l l or by Na ÷ depletion, is the sole factor affecting receptor properties then the two treatments should have given similar results. This view appears not to be supported by our results which instead suggest a more complex mechanism involving electrolytes, as recently proposed 11. A second feature of importance

generated A l l ) the BBB may no longer be valid. This f u n d a m e n t a l change in our view of the accessibility of the A l l receptor areas should lead to a re-evaluation of the 'brain' r e n i n - a n g i o t e n s i n system and its interaction with the peripheral counterpart, and to an increase in interest in the functional role of A l l receptor sites outside the circumventricular organs. ACKNOWLEDGEMENTS

is that all areas, whether 'inside' or 'outside' the BBB, were affected by at least one of the treatments (Table I). W h e t h e r blood-borne A l l reaches areas within the BBB is not clear since short-term uptake studies show the BBB to be impervious to AI129. It is

The assistance of David de Vries and Melissa Little is gratefully acknowledged. This research was supported by a grant from the University of Q u e e n s l a n d Brain Research Fund.

REFERENCES

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