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Characterization of angiotensin II receptors in the anterior pituitary gland
Molecular and Cellular Endocrinology,
Elsevier/North-Holland
25 (1982) 203-212 Scientific Publishers, Ltd.
203
CHARACTERIZATION OF ANGIOTENSIN II RECEPTORS IN THE ANTERIOR PITUITARY GLAND **
R.L. HAUGER *, G. AGUILERA,
A.J. BAUKAL and K.J. CATT
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20205 IU.S.A.)
Received 24 August 1981; accepted 9 October 1981
Specific and high affinity binding sites for angiotensin II were demonstrated in the anterior pituitary gland by binding studies with [ r251]iodoangiotensin II. The binding properties of the pituitary receptors were similar to those of angiotensin II receptors present in the adrenal gland. The concentration of binding sites in rat anterior pituitary (293 f 50 fmoles/mg protein) was less than in the adrenal gland, but was much greater than in smooth muscle. Angiotensin II receptors were identified in the anterior pituitary tissue of mature and immature animals of both sexes, and in species including rat, rabbit and dog. No binding of angiotensin II was detected in posterior pituitary homogenates, or in GH3 pituitary tumor cells. Collagenasedispersed anterior pituitary cells also contained specific binding sites for angiotensin II, with equilibrium binding constant (K,) of 3.6 X lo9 M-l. The presence of specific high-affinity angiotensin II receptor in the anterior pituitary gland provides a mechanism by which angio-
tensin-like peptides could modulate the process of pituitary hormone secretion. Keywords:
angiotensin II; receptors; pituitary.
An increasing number of peptide hormones appear likely to have important roles in the central nervous system (Vale et al., 1977; Snyder, 1980; Emson, 1979). Angiotensin II, which has been established to be a major regulator of aldosterone secretion and vascular smooth muscle contraction (Aguilera et al., 1978; Williams et al., 1976), may also be a significant regulatory peptide in the central nervous system. Isorenin, renin substrate, angiotensin I, angiotensinconverting enzymes and angiotensinase are present in the central nervous system and pituitary gland (Hirose et al., 1978; Ganten et al., 1978; Slater et al., 1980). Also, angiotensin II has been identified in brain and spinal cord neurones, the highest concentrations being detected in the medial external layer of the median eminence, central amygdala, substantia gelatinosa of the spinal cord, spinal nucleus of the trigerninal tract, and sympathetic lateral column (Ganten et al., 1978). Angiotensin II can * Present address: Clinical Psychobiology Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20205 (U.S.A.). ** Address all correspondence to Dr. G. Aguilera. 0303-7207/82/0000-0000/$02.75
0 1982 Elsevier/North-Holland
Scientific Publishers, Ltd.
204
R.L. Hauger et al.
elicit centrally-mediated pressor responses and dipsogenic behaviour, and enhances the release or synthesis of catecholamines and serotonin, and release of vasopressin (Peach, 1977; Ganong, 1977; Fitzsimmons, 1978; Ramsay et al., 1978). These effects can be evoked by agonists and blocked by antagonists of angiotensin II, with activities comparable to the biological potencies of these peptides in the adrenal and smooth muscle (Glossmann et al., 1974; Baudouin et al., 1971). Recently, angiotensin II receptor sites have been demonstrated in areas of the brain associated with these central effects, namely the hypothalamus, organum vasculosum, thalamus, midbrain and brainstem (Bennett and Snyder, 1976; Landas et al., 1980). These several lines of evidence strongly suggest that angiotensin II may be a neurotransmitter in the central nervous system. We now report the presence of specific high-affinity receptor sites for angiotensin II in the anterior pituitary. These sites are less abundant than in the adrenal zona glomerulosa, but are considerably more concentrated than in the hypothalamus or uterine smooth muscle. These findings suggest that angiotensin II may have a significant biological role in the regulation of pituitary function.
MATERIALS
AND METHODS
Synthetic [Asp’, Ile5]-, des-Asp’-, and [Sar’, Ala81 -angiotensin II were obtained from Beckman Instruments, Inc. (Palo Alto, CA) and [Sari]-angiotensin II was a gift from Dr. MC. Khosla, Research Division, Cleveland Clinic, Cleveland, OH. Peptides were dissolved in 0.01 M acetic acid and the concentration was standardized by reference to the ultraviolet absorption of tyrosine. Monoiodinated i2’I-labeled angiotensin II was obtained from New England Nuclear Corp. (Boston, MA). The specific activity of the labeled peptide ranged from 650 to 12OOpCi per pg, as determined by radioimmunoassay and radioligand-receptor assay. Male and female Sprague-Dawley rats (100-200 g) from Charles River, Inc. (Wilmington, MA) were maintained on normal sodium diet and housed in facilities with controlled lighting. Adrenal and uterine muscle homogenates were prepared as described previously (Aguilera et al., 1978; Glossmann et al., 1974a). The posterior pituitary was separated from the anterior pituitary by needle dissection, and each portion of the gland was dispersed in 20 mM sodium bicarbonate by 10 strokes in a teflon-glass homogenizer. Particulate membrane-rich fractions were prepared as described for adrenal gland and uterine smooth muscle, 25 +_3 pituitaries being used for each preparation. Pituitary glands from 6-month-old male beagle dogs and mature New Zealand white female rabbits were rapidly removed after killing the animals with an overdose of pentobarbital. In one experiment, male rats were decapitated, their brains rapidly removed, and hypothalami were dissected out op ice as described previously (Bennett and Snyder, 1976). Whole hypothalamic membrane fractions were prepared as described for the pituitary gland. Protein concentrations were measured by employing bovine serum albumin (BSA) as a standard (Lowry et al.,
Pituitary
receptors for angiotensin
II
205
II to 600-30 000 X g membrane-rich 1951). The binding of [ ‘2sI]angiotensin pellets from the homogenates of anterior and posterior pituitary, and hypothal~us, was determined by a modi~cation of the procedure described for particulate adrenal and muscle fractions (Glossmann et al., 1974a; Aguilera and Catt, 1981). Pituitary membranes (SO-100 lug of protein) were incubated with 0.5 nM [1251] angiotensin II, in a total volume of 2.50 ~1 containing 50 mM Tris-HCl buffer pH 7.4, 10 mM MgC12, 1 mM dithiothreitol, 1 mM EGTA and 0.5% of BSA. Nonspecific binding was determined in the presence of 0.1 PM unlabeled angiotensin II. Incubations were performed at 22*C for 45 min, and the reaction was terminated by rapid filtration on GF/C glass fibre filters. Anterior pituitary cells were prepared by a modification of a previously described method (Vale et al., 1972). Anterior pituitaries (the mean number of anterior pituitaries for each cell dispersion was 52 + 8) were collected from female rats weighing 100-150 g, immediately placed in medium 199, and thoroughly minced with fine scissors. ‘The tissue suspension was then tr~sferred to medium 199 containing 0.35% collagenase (Worthington), 0.1% hyaluronidase (Sigma), 0.05% soybean trypsin inhibitor (Sigma), l-3% bovine serum albumin (Sigma), and 0.001% DNAase (Sigma), and incubated at 37’C for 45 min with constant gentle agitation. The preparation was then pelleted at 100 Xg for 5 min, resuspended in calcium-free medium 199 with l-3% sucrose and 0.2% bovine serum albumin, and again centrifuged at 900 rpm for 5 min. Pituitary cells were then prepared by physical dispersion of the enzyme-treated tissue, by resuspending the pellet in calcium-free medium 199 containing 0.2% bovine serum albumin and 0.001% DNAase, and repeatedly drawing the suspension through 3 mm ID polyethylene tubing attached to a syringe. The harvested cells were immediately diluted in IO-20 vofumes of medium 199 containing 5 mM KC1 and 0.2% bovine serum albumin (incubation medium). The dispersion procedure was accomplished within 20 min. The isolated anterior pituitary cells were then pelleted at 100 Xg for 5 min and resuspended in incubation medium. Binding of [ ‘*‘I]angiotensin 11 to dispersed anterior pituitary cells was measured as previously described for adrenal glomerulosa cells (DougIas et al., 1978), using 3.4 -t 0.5 X lo5 anterior pituita~ cells for each binding dete~ination. For analysis of binding to pituitary tumor cells, GHa cells were obtained from the American Type Culture Collection, Rockville, MD. Viability of the cells was assessed by the trypan blue method to be between 70 and 80%. Receptor binding capacities and association constants (K,) were calculated by computer analysis of the experimental data (Ketelslegers et al., 1975). The angiotensin 11 and peptide concentrations producing 50% in~bition of [ ‘*sI]~~otens~ II binding (ID& were determined by computer analysis using a 4-parameter logistic function (Rodbard and Hutt, 1974).
R.L. Hauger et al.
206
RESULTS Specific binding of angiotensin II to pituitary membranes increased with time and reached a plateau after 45 min. As shown in Fig. 1, the specific binding was 10 times higher than the non-specific uptake measured in the presence of excess unlabelled hormone. At equilibrium, angiotensin II binding to pituitary particles was linear when the protein concentration was increased from 30 to 100 Dg per incubation tube (Fig. 2). No significant binding of angiotensin II was detected in fractions from the posterior pituitary gland. Storage of anterior pituitary tissue at -40°C for 6 days resulted in a 60% decrease in [ “‘1 ] angiotensin II binding. Likewise, elevation of the incubation temperature to 37°C decreased [1251]angiotensin II binding by 20-30% at 45 min, and no apparent steady state was reached. Angiotensin II binding in anterior pituitary particles was enhanced by increasing the concentration of divalent cations (Mg*+ and Ca*‘) in the binding assay from 0 to 10 mM. In the presence of, 10 mM Mg, and EGTA or EDTA at concentrations from 0.1 to 1 mM, the binding of angiotensin II to pituitary membranes was further increased. However, higher concentrations of EDTA (2-10 mM) progressively decreased binding to the levels observed in the absence of cations (Fig. 3). No changes in binding were observed by addition of sodium chloride from 10 to 140 mM. The increases in angiotensin II binding observed in the presence of low concentrations of EDTA and EGTA were due to decreased degradation of the free peptide during the binding assay, as determined by radioimmunoassay of angiotensin II
TlMElminl
Fig. 1. Time course of specific and non-specific pituitary particles. Points are the mean of duplicate
,,g PROTEIN
binding of incubations
[ ’ *‘I] angiotensin II in anterior in 1 of 3 similar experiments.
Fig. 2. Relationship between protein concentration and binding of [ ‘251]angioten~in II to anterior and posterior pituitary particles. Points are the mean of duplicate incubations in 1 of 3 similar experiments.
Pituitary receptors for angiotensin II
207
.
Control 0 MgCI, 10 mM
A CaCI, 10 mM
01
I
I
I
I
I
2
4
6
8
10
EDTA CONCENTRATION
imM)
Fig. 3. Effect of cations, EGTA and EDTA on the binding of [ 1251]angiotensin II to anterior pituitary membranes. Points are the mean of duplicate incubations 1 of 3 similar experiments.
in incubation medium. Similarly, the addition of dithiothreitol (0.1-I mM) enhanced angiotensin II binding to pituitary membranes by preventing degradation of the free angiotensin. Higher concentrations of dithiothreitol in the binding assay markedly decreased angiotensin II binding to pituitary membranes (Fig. 4). Binding of angiotensin II to membrane-rich particles prepared from the rat anterior pituitary gland was saturable and of high affinity. In 13 Expts., the mean binding capacity for angiotensin II was 293 f 50 fmoles/mg protein, and the equilibrium association constant (K,) was 1 .I + 0.2 X 10’ M-r. The binding of angiotensin II to anterior pituitary particles in a typical experiment is shown in Fig. 5, in comparison to the binding observed in adrenal glomerulosa and uterine fractions. In this experiment, the binding capacity of anterior pituitary tissue (262 + 9 fmoles/mg protein) was less than observed in the adrenal zona glomerulosa (1928 + 194 fmoles/mg), but was markedly greater than in uterus (134 + 10 fmoles/mg). The angiotensin II binding capacity of crude hypothalamic tissue was about l/30 of the receptor content measured in the anterior pituitary gland (data not shown). The association constant (KJ for angiotensin II binding in the anterior pituitary (1.8 + 0.03 X 10’ M-r), and somewhat higher than that of uterine smooth muscle (0.25 * 0.04 X 10’ M-r) measured in the same experiment. Angiotensin II receptors were also demonstrated in anterior pituitary glands of dogs and rabbits of both sexes. In the dog, the binding capacity of anterior pituitary homogenate for [1251]angiotensin II was 395 fmoles/mg, and binding affinity (K,) was 4 X 10’ M-i. The rabbit pituitary exhibited lower binding capacity for angiotensin II (32 fmoles/mg) and binding affinity was not determined.
R.L. Hauger et al.
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TOTAL
ANGIOTENSIN
II hM1
Fig. 4. Effect of increasing concentrations of dithiothreitol (DTT) on angiotensin II binding to anterior pituitary particles (bottom panel), and degradation of the free ligand during the binding assay (top panel). Degradation of angiotensin II was determined by radioimmunoassay of angiotensin II in the media after incubation of the peptide with pituitary particles (initial concentration 1 nM). Bars represent the mean of duplicate incubations in 1 of 2 similar experiments. Fig. 5. [ 1251]Angiotensin II binding to adrenal (o), pituitary (o), and uterus (a). 30 adult female rats were used for the experiment. Each point represents the meui of triplicate replications in the binding assay.
In the anterior pituitary, angiotensin II binding was competitively inhibited by agonist derivatives and the antagonist [Sarr, Ala8]angiotensin II, as shown for a typical experiment in Fig. 6. When binding-inhibition activities of the agonist derivatives of angiotensin II were analyzed, the ICse for angiotensin II was 1.5 f 0.3 nM (mean 5 SE, n = 5) and [Sari langiotensin II was the most potent inhibitor of [1251]angiotensin II binding, with ICse of 0.28 nM. The heptapeptide des-Asp’angiotensin II was relatively less active than angiotensin II, with I&e of 10.3 + 2.2 r&l (n = S), and [Sar’, Ala*]angiotensin II exhibited an IDS0 of 1.1 + 0.1 nM (n = 2). Isolated pituitary cells prepared by collagenase dispersion of anterior pituitary II. Scatchard analysis of glands also exhibited specific binding of [ r2’I]angiotensin binding data, shown for a representative experiment in Fig. 7, demonstrated that isolated pituitary cells contain a single population of angiotensin binding sites, as did the anterior pituitary particles. The mean angiotensin II binding capacity of anterior pituitary cells in 8 experiments was 22.9 + 2.8 fmoles per IO6 cells, and the mean association constant was 3.6 f 0.4 X 10’ M-l (n = 8). Similar values were obtained in acutely dispersed cells and those incubated overnight prior to angio-
Pituitary receptors for angiotensin II
01 lo-" ’
I
lo-'0
I
209
I
I
10-g 10-o
I
10-7 0
PEPTIDE CONCENTRATION
(Ml
100
BOUND ANGIOTENSIN
200 II Ifmoles/mgi
Fig. 6. Displacement of [ 1z 5 I] angiotensin II from anterior pituitary membranes by angiotensin II (e), [Sar’, AlaaJangiotensin II (o), [Sar’langiotensin II (b), and des-Aspr-angiotensin II (0). 31 anterior pituitary glands were used in the experiment, and binding-inhibition studies were performed as previously described with 6+7 X 10-l’ M [ 1251]angiotensin II and increasing concentrations of unlabeled peptide from 10-r’ to lo-’ M. Each point represents the mean of triplicate determinations in the binding assay. The IDso values calculated by computer analysis using a 4-parameter logistic function for angiotensin II, [Sari, Ala8]angiotensin II, and des-Asp’-angiotensin II were 2.7, 1.2 and 13.4 nM, respectively. Fig. 7. [ I2 SI]Angiotensin 11 binding to collagenase-dispersed anterior pituitary cells. In this representative experiment, anterior pituitary glands were obtained from 23 female rats and prepared as described in Materials and Methods. Each point indicates the mean of triplicate determinations in the binding assay. The binding capacity was 37.0 f 1.6 fmoles per lo6 cells and the association constant (Ka) was 3.2 k 0.5 X 10’ M-r, as calculated by computer analysis of the binding data (Ketelslegers et al., 1975). Inset, Scatchard analysis of [l 251]angiotensin II binding to collagenase-dispersed anterior pituitary cells. The binding capacity was 37.6 * 1.8 fmoles per lo6 cells and K, was 3.0 1 0.4 M-‘.
tensin II binding assay. No appreciable binding of angiotensin II was detected in GHa pituitary tumor cells (data not shown). In preliminary studies designed to explore the interaction of angiotensin II with specific cell types, no actions of the octapeptide on release of ACTH or LH were detected in isolated pituitary cells. The effects on ACTH release were evaluated in cells prepared as described above, with assay of medium ACTH in isolated adrenal fasciculata cells (Douglas et al., 1978). The effects of angiotensin II on LB release were examined in 2-day cultured pituitary cells that released LH in response to LHRH in the same experiment (Conn et al., 1979). In such cells, angiotensin II in concentrations up to IO-’ M did not cause LH release, and did not inhibit the LH release evoked by 10m6 M GnRH.
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DISCUSSION These studies have demonstrated that specific binding sites with high affinity for angiotensin II are present in relatively high concentration in the anterior pituitary gland. The peptide specificity and binding affinity of the pituitary receptors for angiotensin II were similar to those of adrenal glomerulosa cells (Glossmann et al., 1974a), and smooth muscle (Baudouin et al., 1971) 2 of the biologically important sites of action of angiotensin II, and to those of receptors present in the brain (Bennett and Snyder, 1976). In contrast to the adrenal glomerulosa in which angiotensin II binding is enhanced by sodium (Glossmann et al., 1974b; Douglas et al., 1978), the binding of the peptide to pituitary membranes was independent of the sodium concentration. However, binding to pituitary membranes was increased in the presence of the divalent cations Mg, Mn and Ca, as previously shown for angiotensin II binding to the smooth muscle receptor (Aguilera and Catt, 1981). A corrrrtm-pr&m -iIs the-Muant~Qf peptideharmane binding to particulate cell fractions is the degradation of the free ligand during the binding assay. Breakdown of free angiotensin II has been partially prevented by the use of low concentrations of EDTA and dithiothreitol in the incubation (Glossmann et al., 1974a; Baukal and Aguilera, 1980). Even though concentrations of dithiothreitol higher than 1 mM are more effective in preventing degradation (Fig. 4), angiotensin II binding to the pituitary membranes was markedly decreased, indicating that disulfide bonds are necessary for the functional integrity of the angiotensin II receptor. The slightly lower affinities measured in muscle homogenates could result from greater degradation of angiotensin II in those tissues than in the adrenal gland. Also, the lower apparent binding affinity of the pituitary receptors for the desAsp’-heptapeptide than for angiotensin II is probably due to more rapid metabolism of the heptapeptide, as recently shown in rat adrenal cells (Aguilera et al., 1979). Specific binding sites for angiotensin II have been identified in the thalamushypothalamus, midbrain, and organum vasculosum, areas which appear to be involved in mediating the central actions of angiotensin II (Bennett and Snyder, 1976; Landas et al., 1980). In the present study, the concentration of angiotensin II receptor sites in the anterior pituitary was considerably greater than in the hypothalamus, and in a recognized target cell such as smooth muscle. Pituitary binding of angiotensin II was also noted in a recent report in which uptake of [1251]angiotensin I and [r4C]angiotensin II was determined in vivo (Gregg and Malvin, 1977). However, the posterior pituitary accumulated slightly more labeled angiotensin than the anterior pituitary, whereas we did not detect any specific binding of [‘251]angiotensin II to posterior pituitary homogenates in the present experiments. Another recent report on the distribution of angiotensih II binding in rat brain (Sirett et al., 1977) noted specific hypothalamic binding (11 -L2 fmoles/mg protein), similar to that observed in the present study (9 fmoles/mg), with relatively high binding in the anterior pituitary and no uptake in the posterior pituitary.
Pituitary receptors for angiotensin II
211
Numerous central nervous system peptides have been localized to the hypothalamus and may function as neurotransmitters. Also, these hypothalamic peptides may be released into the portal vessels and subsequently regulate anterior pituitary secretion similar to the ‘classical’ releasing factors. Enkephalins, gastrins and neurotensin have been reported to influence anterior pituitary secretion (Krulich, 1979). Since the anterior pituitary contains measurable amounts of both enkephalins and gastrins, as well as binding sites for opiates and neurotensin (Vijayan et al., 1978; Rivier et al., 1977; Tramy and Leonardeu, 1979; Rehfeld, 1978; Lazarus et al., 1977), these peptides may exert a direct action on pituitary function. The existence of a fairly large number of angiotensin II-positive nerve terminals in the medial external layer of the median eminence has suggested that angiotensin II may be involved in control of hormone secretion from the anterior pituitary, either by release as a hypothalamic hormone or by influencing the secretion of other regulatory hormones from the median eminence. The present finding that specific high-affinity receptor sites for angiotensin II are abundant in the anterior pituitary suggests that angiotensin II may be released from the median eminence and bind to anterior pituitary cells to modulate their secretory functions. Since angiotensin II receptors are not present in the posterior pituitary, the well-recognized actions of angiotensin II on vasopressin secretion would occur by the interaction of angiotensin II with extrapituitary receptors, probably at the hypothalamic level. Although an exact role for angiotensin II in the anterior pituitary has yet to be identified, there is preliminary evidence for an action of angiotensin II upon pituitary hormone release. It has been shown that concentrations of angiotensin II in the micromolar range caused a small increase in luteinizing hormone, growth hormone and prolactin release when incubated with hemipituitaries in vitro, whereas intraventricular infusion of the peptide decreased circulating prolactin and growth hormone (Steele et al., 1981). Recent studies in our laboratory demonstrated a direct stimulatory effect of angiotensin II on prolactin release in collagenase-dispersed rat pituitary cells (Aguilera et al., unpublished). This effect was observed with nanomolar concentrations of angiotensin 11,suggesting that the actions of the peptide in the anterior pituitary are mediated by the angiotensin II receptors described in the present report, Other possible target cells for angiotensin in the anterior pituitary are corticotrophs, since release of ACTH by angiotensin II is wellestablished (Ramsay et al., 1978). This effect of angiotensin II has been attributed to a direct action on the pituitary gland (Maran and Yates, 1978), though other data (Gann, 1969) is more consistent with an indirect action on ACTH secretion via stimulation of the medium eminence. In the present experiments, using rat fasciculata cells as a bioassay for corticotropin, it was not possible to detect any increase in ACTH release by angiotensin II in collagenase-dispersed pituitary cells. However, there is the possibility that more sensitive approaches could reveal a direct action of angiotensin on corticotrophin secretion. The present study has demonstrated a high concentration of specific receptor sites for angiotensin II in the adenohypophysis, and provides a basis for more detailed investigation of the role of angiotensin II in regulation of pituitary hormone secretion.
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al.
REFERENCES Aguilera, G., and Catt, K.J. (1981) Circ. Res. 49, 751-758. Aguilera, G., Hauger, B.L., and Catt, K.J. (1978) Proc. Natl. Acad. Sci. (U.S.A.) 7.5, 975-979. Aguilera, G., Capponi, A., Baukal, A., Fujita, K., Hauger, R., and Catt, K.J. (1979) Endocrinology 104,1279-1285. Baudot&, M., Meyer, P., and Worcel, M. (1971) Biochem. Biophys. Res. Commun. 42, 434440. Baukal, A., and Aguilera, G. (1980) Endocrine Sot. U.S.A. 62nd Atmual Meeting, Abstract 374. Bennett, J.P., and Snyder, S.H. (1976) .I. Biol, Chem. 251, 7423-7430. Corm, P.M., More& D.V., Dufau, M.L., and Catt, K.J. (1979) Endocrinology 104,448-453. Douglas, J., Aguilera, G., Kondo, T., and Catt, K.J. (1978) Endocrinology 102,685-696. Emson, P.C. (1979) Progr. Neurobiol. 13,61-116. Fitz~mmons, J.T. (1978) Fed. Proc. 37,2669-2675. Gann, D.S. (1969) Ann. N.Y. Acad. Sci. 156,740-7X Ganong, W.F. (1977) Fed. Proc. 36,1771-1775. Ganten, D., Fuxe, K., Phillips, MI., Mann J.F.E., and Ganten, U. (1978) in: Frontiers in Neuroendocrinology, Eds, W.F. Ganong and L. Martini, (Raven, New Y ark) pp. 6 l-99. Glossmann, H., Baukal, A., and Catt, K.J. (1974a) J. Biol. Chem. 249, 82S-834. Glossmann, H., Baukal, A., and Catt, K.J. (1974b) Science l&5,281-283. Gregg, C.M., and Malvin, R.L. (1977) Proc. Sot. Exp. Biol. Med. 155,400-404. Hirose, S., Yokosawa, H., and Inagami, T. (1978) Nature (London) 274, 392-393. Ketelslegers, J.-M., Knott, C.D., and Catt, K.J. (1975) Biochemistry 14, 3075-3083. Krulich, L. (1979) Annu. Rev. physiol. 41,603-615. Landas, S., Phillips, MI., Stamler, J.F., and Raizada, M.K. (1980) Science 210, 791-393. Lazarus, L.H., Brown, M.R., and Perrin, M.H. (1977) Neuropharmacology 16,614-629. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Cbem. 193, 265275. Maran, J.F., and Yates, F.E. (1978) Am. J. Physiol. 233, E273-E285. Peach, M.J. (1977) Physiol. Rev. 57,313-370. Ramsay, D.J., Keil, L.C., Sharpe, M.C., and Shonsalco, J. (1978) Am. J. Physiol. 234, R66R71. Rehfeld, J.F. (1978) Nature (London) 271, 771-773. Rivier, C., Brown, M., and Vale, W. (1977) Endocrinology 100,751-754. Rodbard, D., and Hutt, D. (1974) in: Radioimmunoassay and Related Procedures in Medicine (International Atomic Energy Agency, Vienna) pp. 165. Sirett, N.P., McLean, A.S., Bray, SS., and Hubbard, J.I. (1977) Brain Res. 122, 299-312. Slater, E.E., Defendon, R., and Zimmermann, E.A. (1980) Proc. Natl. Acad. Sci. (U.S.A.) 77, 5458-5460. Snyder, S.H. (1980) Science 209,976-983. Steele, M.K., Negro-Viiar, A., and McCann, S.M. (1981) Endocrinology 109,893-899. Tramy, G., and Leonardeu, J. (1979) Brain Res. 168,457-471. Vale, W., Grant. G., Amoss, M., Blackwell, R., and Guillemin, R. (1972) Endocrinology 91, 562-572. Vale, W., Rivier, C., and Brown. M. (1977) Annu. Rev. Physiol. 39,47S-527. Vijayan, E., Samson, W.K., and McCann, SM. (1978) Life Sci. 23, 2225-2232. WiiUiams, G.H:, HolIerberg, N.K., and Braley, L.M. (1976) Endocrinology P&1343-1350.
Elsevier/North-Holland
25 (1982) 203-212 Scientific Publishers, Ltd.
203
CHARACTERIZATION OF ANGIOTENSIN II RECEPTORS IN THE ANTERIOR PITUITARY GLAND **
R.L. HAUGER *, G. AGUILERA,
A.J. BAUKAL and K.J. CATT
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20205 IU.S.A.)
Received 24 August 1981; accepted 9 October 1981
Specific and high affinity binding sites for angiotensin II were demonstrated in the anterior pituitary gland by binding studies with [ r251]iodoangiotensin II. The binding properties of the pituitary receptors were similar to those of angiotensin II receptors present in the adrenal gland. The concentration of binding sites in rat anterior pituitary (293 f 50 fmoles/mg protein) was less than in the adrenal gland, but was much greater than in smooth muscle. Angiotensin II receptors were identified in the anterior pituitary tissue of mature and immature animals of both sexes, and in species including rat, rabbit and dog. No binding of angiotensin II was detected in posterior pituitary homogenates, or in GH3 pituitary tumor cells. Collagenasedispersed anterior pituitary cells also contained specific binding sites for angiotensin II, with equilibrium binding constant (K,) of 3.6 X lo9 M-l. The presence of specific high-affinity angiotensin II receptor in the anterior pituitary gland provides a mechanism by which angio-
tensin-like peptides could modulate the process of pituitary hormone secretion. Keywords:
angiotensin II; receptors; pituitary.
An increasing number of peptide hormones appear likely to have important roles in the central nervous system (Vale et al., 1977; Snyder, 1980; Emson, 1979). Angiotensin II, which has been established to be a major regulator of aldosterone secretion and vascular smooth muscle contraction (Aguilera et al., 1978; Williams et al., 1976), may also be a significant regulatory peptide in the central nervous system. Isorenin, renin substrate, angiotensin I, angiotensinconverting enzymes and angiotensinase are present in the central nervous system and pituitary gland (Hirose et al., 1978; Ganten et al., 1978; Slater et al., 1980). Also, angiotensin II has been identified in brain and spinal cord neurones, the highest concentrations being detected in the medial external layer of the median eminence, central amygdala, substantia gelatinosa of the spinal cord, spinal nucleus of the trigerninal tract, and sympathetic lateral column (Ganten et al., 1978). Angiotensin II can * Present address: Clinical Psychobiology Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20205 (U.S.A.). ** Address all correspondence to Dr. G. Aguilera. 0303-7207/82/0000-0000/$02.75
0 1982 Elsevier/North-Holland
Scientific Publishers, Ltd.
204
R.L. Hauger et al.
elicit centrally-mediated pressor responses and dipsogenic behaviour, and enhances the release or synthesis of catecholamines and serotonin, and release of vasopressin (Peach, 1977; Ganong, 1977; Fitzsimmons, 1978; Ramsay et al., 1978). These effects can be evoked by agonists and blocked by antagonists of angiotensin II, with activities comparable to the biological potencies of these peptides in the adrenal and smooth muscle (Glossmann et al., 1974; Baudouin et al., 1971). Recently, angiotensin II receptor sites have been demonstrated in areas of the brain associated with these central effects, namely the hypothalamus, organum vasculosum, thalamus, midbrain and brainstem (Bennett and Snyder, 1976; Landas et al., 1980). These several lines of evidence strongly suggest that angiotensin II may be a neurotransmitter in the central nervous system. We now report the presence of specific high-affinity receptor sites for angiotensin II in the anterior pituitary. These sites are less abundant than in the adrenal zona glomerulosa, but are considerably more concentrated than in the hypothalamus or uterine smooth muscle. These findings suggest that angiotensin II may have a significant biological role in the regulation of pituitary function.
MATERIALS
AND METHODS
Synthetic [Asp’, Ile5]-, des-Asp’-, and [Sar’, Ala81 -angiotensin II were obtained from Beckman Instruments, Inc. (Palo Alto, CA) and [Sari]-angiotensin II was a gift from Dr. MC. Khosla, Research Division, Cleveland Clinic, Cleveland, OH. Peptides were dissolved in 0.01 M acetic acid and the concentration was standardized by reference to the ultraviolet absorption of tyrosine. Monoiodinated i2’I-labeled angiotensin II was obtained from New England Nuclear Corp. (Boston, MA). The specific activity of the labeled peptide ranged from 650 to 12OOpCi per pg, as determined by radioimmunoassay and radioligand-receptor assay. Male and female Sprague-Dawley rats (100-200 g) from Charles River, Inc. (Wilmington, MA) were maintained on normal sodium diet and housed in facilities with controlled lighting. Adrenal and uterine muscle homogenates were prepared as described previously (Aguilera et al., 1978; Glossmann et al., 1974a). The posterior pituitary was separated from the anterior pituitary by needle dissection, and each portion of the gland was dispersed in 20 mM sodium bicarbonate by 10 strokes in a teflon-glass homogenizer. Particulate membrane-rich fractions were prepared as described for adrenal gland and uterine smooth muscle, 25 +_3 pituitaries being used for each preparation. Pituitary glands from 6-month-old male beagle dogs and mature New Zealand white female rabbits were rapidly removed after killing the animals with an overdose of pentobarbital. In one experiment, male rats were decapitated, their brains rapidly removed, and hypothalami were dissected out op ice as described previously (Bennett and Snyder, 1976). Whole hypothalamic membrane fractions were prepared as described for the pituitary gland. Protein concentrations were measured by employing bovine serum albumin (BSA) as a standard (Lowry et al.,
Pituitary
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II
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II to 600-30 000 X g membrane-rich 1951). The binding of [ ‘2sI]angiotensin pellets from the homogenates of anterior and posterior pituitary, and hypothal~us, was determined by a modi~cation of the procedure described for particulate adrenal and muscle fractions (Glossmann et al., 1974a; Aguilera and Catt, 1981). Pituitary membranes (SO-100 lug of protein) were incubated with 0.5 nM [1251] angiotensin II, in a total volume of 2.50 ~1 containing 50 mM Tris-HCl buffer pH 7.4, 10 mM MgC12, 1 mM dithiothreitol, 1 mM EGTA and 0.5% of BSA. Nonspecific binding was determined in the presence of 0.1 PM unlabeled angiotensin II. Incubations were performed at 22*C for 45 min, and the reaction was terminated by rapid filtration on GF/C glass fibre filters. Anterior pituitary cells were prepared by a modification of a previously described method (Vale et al., 1972). Anterior pituitaries (the mean number of anterior pituitaries for each cell dispersion was 52 + 8) were collected from female rats weighing 100-150 g, immediately placed in medium 199, and thoroughly minced with fine scissors. ‘The tissue suspension was then tr~sferred to medium 199 containing 0.35% collagenase (Worthington), 0.1% hyaluronidase (Sigma), 0.05% soybean trypsin inhibitor (Sigma), l-3% bovine serum albumin (Sigma), and 0.001% DNAase (Sigma), and incubated at 37’C for 45 min with constant gentle agitation. The preparation was then pelleted at 100 Xg for 5 min, resuspended in calcium-free medium 199 with l-3% sucrose and 0.2% bovine serum albumin, and again centrifuged at 900 rpm for 5 min. Pituitary cells were then prepared by physical dispersion of the enzyme-treated tissue, by resuspending the pellet in calcium-free medium 199 containing 0.2% bovine serum albumin and 0.001% DNAase, and repeatedly drawing the suspension through 3 mm ID polyethylene tubing attached to a syringe. The harvested cells were immediately diluted in IO-20 vofumes of medium 199 containing 5 mM KC1 and 0.2% bovine serum albumin (incubation medium). The dispersion procedure was accomplished within 20 min. The isolated anterior pituitary cells were then pelleted at 100 Xg for 5 min and resuspended in incubation medium. Binding of [ ‘*‘I]angiotensin 11 to dispersed anterior pituitary cells was measured as previously described for adrenal glomerulosa cells (DougIas et al., 1978), using 3.4 -t 0.5 X lo5 anterior pituita~ cells for each binding dete~ination. For analysis of binding to pituitary tumor cells, GHa cells were obtained from the American Type Culture Collection, Rockville, MD. Viability of the cells was assessed by the trypan blue method to be between 70 and 80%. Receptor binding capacities and association constants (K,) were calculated by computer analysis of the experimental data (Ketelslegers et al., 1975). The angiotensin 11 and peptide concentrations producing 50% in~bition of [ ‘*sI]~~otens~ II binding (ID& were determined by computer analysis using a 4-parameter logistic function (Rodbard and Hutt, 1974).
R.L. Hauger et al.
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RESULTS Specific binding of angiotensin II to pituitary membranes increased with time and reached a plateau after 45 min. As shown in Fig. 1, the specific binding was 10 times higher than the non-specific uptake measured in the presence of excess unlabelled hormone. At equilibrium, angiotensin II binding to pituitary particles was linear when the protein concentration was increased from 30 to 100 Dg per incubation tube (Fig. 2). No significant binding of angiotensin II was detected in fractions from the posterior pituitary gland. Storage of anterior pituitary tissue at -40°C for 6 days resulted in a 60% decrease in [ “‘1 ] angiotensin II binding. Likewise, elevation of the incubation temperature to 37°C decreased [1251]angiotensin II binding by 20-30% at 45 min, and no apparent steady state was reached. Angiotensin II binding in anterior pituitary particles was enhanced by increasing the concentration of divalent cations (Mg*+ and Ca*‘) in the binding assay from 0 to 10 mM. In the presence of, 10 mM Mg, and EGTA or EDTA at concentrations from 0.1 to 1 mM, the binding of angiotensin II to pituitary membranes was further increased. However, higher concentrations of EDTA (2-10 mM) progressively decreased binding to the levels observed in the absence of cations (Fig. 3). No changes in binding were observed by addition of sodium chloride from 10 to 140 mM. The increases in angiotensin II binding observed in the presence of low concentrations of EDTA and EGTA were due to decreased degradation of the free peptide during the binding assay, as determined by radioimmunoassay of angiotensin II
TlMElminl
Fig. 1. Time course of specific and non-specific pituitary particles. Points are the mean of duplicate
,,g PROTEIN
binding of incubations
[ ’ *‘I] angiotensin II in anterior in 1 of 3 similar experiments.
Fig. 2. Relationship between protein concentration and binding of [ ‘251]angioten~in II to anterior and posterior pituitary particles. Points are the mean of duplicate incubations in 1 of 3 similar experiments.
Pituitary receptors for angiotensin II
207
.
Control 0 MgCI, 10 mM
A CaCI, 10 mM
01
I
I
I
I
I
2
4
6
8
10
EDTA CONCENTRATION
imM)
Fig. 3. Effect of cations, EGTA and EDTA on the binding of [ 1251]angiotensin II to anterior pituitary membranes. Points are the mean of duplicate incubations 1 of 3 similar experiments.
in incubation medium. Similarly, the addition of dithiothreitol (0.1-I mM) enhanced angiotensin II binding to pituitary membranes by preventing degradation of the free angiotensin. Higher concentrations of dithiothreitol in the binding assay markedly decreased angiotensin II binding to pituitary membranes (Fig. 4). Binding of angiotensin II to membrane-rich particles prepared from the rat anterior pituitary gland was saturable and of high affinity. In 13 Expts., the mean binding capacity for angiotensin II was 293 f 50 fmoles/mg protein, and the equilibrium association constant (K,) was 1 .I + 0.2 X 10’ M-r. The binding of angiotensin II to anterior pituitary particles in a typical experiment is shown in Fig. 5, in comparison to the binding observed in adrenal glomerulosa and uterine fractions. In this experiment, the binding capacity of anterior pituitary tissue (262 + 9 fmoles/mg protein) was less than observed in the adrenal zona glomerulosa (1928 + 194 fmoles/mg), but was markedly greater than in uterus (134 + 10 fmoles/mg). The angiotensin II binding capacity of crude hypothalamic tissue was about l/30 of the receptor content measured in the anterior pituitary gland (data not shown). The association constant (KJ for angiotensin II binding in the anterior pituitary (1.8 + 0.03 X 10’ M-r), and somewhat higher than that of uterine smooth muscle (0.25 * 0.04 X 10’ M-r) measured in the same experiment. Angiotensin II receptors were also demonstrated in anterior pituitary glands of dogs and rabbits of both sexes. In the dog, the binding capacity of anterior pituitary homogenate for [1251]angiotensin II was 395 fmoles/mg, and binding affinity (K,) was 4 X 10’ M-i. The rabbit pituitary exhibited lower binding capacity for angiotensin II (32 fmoles/mg) and binding affinity was not determined.
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TOTAL
ANGIOTENSIN
II hM1
Fig. 4. Effect of increasing concentrations of dithiothreitol (DTT) on angiotensin II binding to anterior pituitary particles (bottom panel), and degradation of the free ligand during the binding assay (top panel). Degradation of angiotensin II was determined by radioimmunoassay of angiotensin II in the media after incubation of the peptide with pituitary particles (initial concentration 1 nM). Bars represent the mean of duplicate incubations in 1 of 2 similar experiments. Fig. 5. [ 1251]Angiotensin II binding to adrenal (o), pituitary (o), and uterus (a). 30 adult female rats were used for the experiment. Each point represents the meui of triplicate replications in the binding assay.
In the anterior pituitary, angiotensin II binding was competitively inhibited by agonist derivatives and the antagonist [Sarr, Ala8]angiotensin II, as shown for a typical experiment in Fig. 6. When binding-inhibition activities of the agonist derivatives of angiotensin II were analyzed, the ICse for angiotensin II was 1.5 f 0.3 nM (mean 5 SE, n = 5) and [Sari langiotensin II was the most potent inhibitor of [1251]angiotensin II binding, with ICse of 0.28 nM. The heptapeptide des-Asp’angiotensin II was relatively less active than angiotensin II, with I&e of 10.3 + 2.2 r&l (n = S), and [Sar’, Ala*]angiotensin II exhibited an IDS0 of 1.1 + 0.1 nM (n = 2). Isolated pituitary cells prepared by collagenase dispersion of anterior pituitary II. Scatchard analysis of glands also exhibited specific binding of [ r2’I]angiotensin binding data, shown for a representative experiment in Fig. 7, demonstrated that isolated pituitary cells contain a single population of angiotensin binding sites, as did the anterior pituitary particles. The mean angiotensin II binding capacity of anterior pituitary cells in 8 experiments was 22.9 + 2.8 fmoles per IO6 cells, and the mean association constant was 3.6 f 0.4 X 10’ M-l (n = 8). Similar values were obtained in acutely dispersed cells and those incubated overnight prior to angio-
Pituitary receptors for angiotensin II
01 lo-" ’
I
lo-'0
I
209
I
I
10-g 10-o
I
10-7 0
PEPTIDE CONCENTRATION
(Ml
100
BOUND ANGIOTENSIN
200 II Ifmoles/mgi
Fig. 6. Displacement of [ 1z 5 I] angiotensin II from anterior pituitary membranes by angiotensin II (e), [Sar’, AlaaJangiotensin II (o), [Sar’langiotensin II (b), and des-Aspr-angiotensin II (0). 31 anterior pituitary glands were used in the experiment, and binding-inhibition studies were performed as previously described with 6+7 X 10-l’ M [ 1251]angiotensin II and increasing concentrations of unlabeled peptide from 10-r’ to lo-’ M. Each point represents the mean of triplicate determinations in the binding assay. The IDso values calculated by computer analysis using a 4-parameter logistic function for angiotensin II, [Sari, Ala8]angiotensin II, and des-Asp’-angiotensin II were 2.7, 1.2 and 13.4 nM, respectively. Fig. 7. [ I2 SI]Angiotensin 11 binding to collagenase-dispersed anterior pituitary cells. In this representative experiment, anterior pituitary glands were obtained from 23 female rats and prepared as described in Materials and Methods. Each point indicates the mean of triplicate determinations in the binding assay. The binding capacity was 37.0 f 1.6 fmoles per lo6 cells and the association constant (Ka) was 3.2 k 0.5 X 10’ M-r, as calculated by computer analysis of the binding data (Ketelslegers et al., 1975). Inset, Scatchard analysis of [l 251]angiotensin II binding to collagenase-dispersed anterior pituitary cells. The binding capacity was 37.6 * 1.8 fmoles per lo6 cells and K, was 3.0 1 0.4 M-‘.
tensin II binding assay. No appreciable binding of angiotensin II was detected in GHa pituitary tumor cells (data not shown). In preliminary studies designed to explore the interaction of angiotensin II with specific cell types, no actions of the octapeptide on release of ACTH or LH were detected in isolated pituitary cells. The effects on ACTH release were evaluated in cells prepared as described above, with assay of medium ACTH in isolated adrenal fasciculata cells (Douglas et al., 1978). The effects of angiotensin II on LB release were examined in 2-day cultured pituitary cells that released LH in response to LHRH in the same experiment (Conn et al., 1979). In such cells, angiotensin II in concentrations up to IO-’ M did not cause LH release, and did not inhibit the LH release evoked by 10m6 M GnRH.
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DISCUSSION These studies have demonstrated that specific binding sites with high affinity for angiotensin II are present in relatively high concentration in the anterior pituitary gland. The peptide specificity and binding affinity of the pituitary receptors for angiotensin II were similar to those of adrenal glomerulosa cells (Glossmann et al., 1974a), and smooth muscle (Baudouin et al., 1971) 2 of the biologically important sites of action of angiotensin II, and to those of receptors present in the brain (Bennett and Snyder, 1976). In contrast to the adrenal glomerulosa in which angiotensin II binding is enhanced by sodium (Glossmann et al., 1974b; Douglas et al., 1978), the binding of the peptide to pituitary membranes was independent of the sodium concentration. However, binding to pituitary membranes was increased in the presence of the divalent cations Mg, Mn and Ca, as previously shown for angiotensin II binding to the smooth muscle receptor (Aguilera and Catt, 1981). A corrrrtm-pr&m -iIs the-Muant~Qf peptideharmane binding to particulate cell fractions is the degradation of the free ligand during the binding assay. Breakdown of free angiotensin II has been partially prevented by the use of low concentrations of EDTA and dithiothreitol in the incubation (Glossmann et al., 1974a; Baukal and Aguilera, 1980). Even though concentrations of dithiothreitol higher than 1 mM are more effective in preventing degradation (Fig. 4), angiotensin II binding to the pituitary membranes was markedly decreased, indicating that disulfide bonds are necessary for the functional integrity of the angiotensin II receptor. The slightly lower affinities measured in muscle homogenates could result from greater degradation of angiotensin II in those tissues than in the adrenal gland. Also, the lower apparent binding affinity of the pituitary receptors for the desAsp’-heptapeptide than for angiotensin II is probably due to more rapid metabolism of the heptapeptide, as recently shown in rat adrenal cells (Aguilera et al., 1979). Specific binding sites for angiotensin II have been identified in the thalamushypothalamus, midbrain, and organum vasculosum, areas which appear to be involved in mediating the central actions of angiotensin II (Bennett and Snyder, 1976; Landas et al., 1980). In the present study, the concentration of angiotensin II receptor sites in the anterior pituitary was considerably greater than in the hypothalamus, and in a recognized target cell such as smooth muscle. Pituitary binding of angiotensin II was also noted in a recent report in which uptake of [1251]angiotensin I and [r4C]angiotensin II was determined in vivo (Gregg and Malvin, 1977). However, the posterior pituitary accumulated slightly more labeled angiotensin than the anterior pituitary, whereas we did not detect any specific binding of [‘251]angiotensin II to posterior pituitary homogenates in the present experiments. Another recent report on the distribution of angiotensih II binding in rat brain (Sirett et al., 1977) noted specific hypothalamic binding (11 -L2 fmoles/mg protein), similar to that observed in the present study (9 fmoles/mg), with relatively high binding in the anterior pituitary and no uptake in the posterior pituitary.
Pituitary receptors for angiotensin II
211
Numerous central nervous system peptides have been localized to the hypothalamus and may function as neurotransmitters. Also, these hypothalamic peptides may be released into the portal vessels and subsequently regulate anterior pituitary secretion similar to the ‘classical’ releasing factors. Enkephalins, gastrins and neurotensin have been reported to influence anterior pituitary secretion (Krulich, 1979). Since the anterior pituitary contains measurable amounts of both enkephalins and gastrins, as well as binding sites for opiates and neurotensin (Vijayan et al., 1978; Rivier et al., 1977; Tramy and Leonardeu, 1979; Rehfeld, 1978; Lazarus et al., 1977), these peptides may exert a direct action on pituitary function. The existence of a fairly large number of angiotensin II-positive nerve terminals in the medial external layer of the median eminence has suggested that angiotensin II may be involved in control of hormone secretion from the anterior pituitary, either by release as a hypothalamic hormone or by influencing the secretion of other regulatory hormones from the median eminence. The present finding that specific high-affinity receptor sites for angiotensin II are abundant in the anterior pituitary suggests that angiotensin II may be released from the median eminence and bind to anterior pituitary cells to modulate their secretory functions. Since angiotensin II receptors are not present in the posterior pituitary, the well-recognized actions of angiotensin II on vasopressin secretion would occur by the interaction of angiotensin II with extrapituitary receptors, probably at the hypothalamic level. Although an exact role for angiotensin II in the anterior pituitary has yet to be identified, there is preliminary evidence for an action of angiotensin II upon pituitary hormone release. It has been shown that concentrations of angiotensin II in the micromolar range caused a small increase in luteinizing hormone, growth hormone and prolactin release when incubated with hemipituitaries in vitro, whereas intraventricular infusion of the peptide decreased circulating prolactin and growth hormone (Steele et al., 1981). Recent studies in our laboratory demonstrated a direct stimulatory effect of angiotensin II on prolactin release in collagenase-dispersed rat pituitary cells (Aguilera et al., unpublished). This effect was observed with nanomolar concentrations of angiotensin 11,suggesting that the actions of the peptide in the anterior pituitary are mediated by the angiotensin II receptors described in the present report, Other possible target cells for angiotensin in the anterior pituitary are corticotrophs, since release of ACTH by angiotensin II is wellestablished (Ramsay et al., 1978). This effect of angiotensin II has been attributed to a direct action on the pituitary gland (Maran and Yates, 1978), though other data (Gann, 1969) is more consistent with an indirect action on ACTH secretion via stimulation of the medium eminence. In the present experiments, using rat fasciculata cells as a bioassay for corticotropin, it was not possible to detect any increase in ACTH release by angiotensin II in collagenase-dispersed pituitary cells. However, there is the possibility that more sensitive approaches could reveal a direct action of angiotensin on corticotrophin secretion. The present study has demonstrated a high concentration of specific receptor sites for angiotensin II in the adenohypophysis, and provides a basis for more detailed investigation of the role of angiotensin II in regulation of pituitary hormone secretion.
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al.
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