INTRODUCTION

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ANGIOTENSIN II (ANG II), the active octapeptide hormone of the brain renin-angiotensin system (RAS), can have profound actions on many of the systems that make up the hypothalamic-pituitary (HP) axis (12, 13). ANG II has a gonadal steroid-dependent modulation of gonadotropin-releasing hormone (GnRH) (39) release with a subsequent change in pituitary luteinizing hormone (LH) release (12, 38). This control is likely mediated through activation of ANG II receptors in the preoptic-anterior hypothalamic area and the ventral portion of the bed nucleus of the stria terminalis (BSTV) (16, 38). ANG II is believed to regulate fluid and electrolyte balance through activation of ANG II receptors in the subfornical organ (SFO) (25, 28), a function that may also involve an angiotensinergic projection to the paraventricular nucleus of the hypothalamus (PVH) (27). ANG II also plays an important role in stress responses through activation of receptors within the PVH that stimulate corticotropin-releasing hormone (CRH) release and mRNA expression (3, 40), as well as activation of the autonomic division of the PVH, resulting in an increased sympathetic outflow (19). Finally, ANG II stimulates dopamine (DA) release into the portal blood system through activation of receptors on dopaminergic neurons and nerve terminals within the arcuate nucleus (ARH) and median eminence (ME), respectively, resulting in inhibition of prolactin (Prl) release (20, 29).

The state of lactation is characterized by dramatic changes in the HP axis. The lactating rat has suckling-induced anovulation, resulting from near-undetectable levels of LH release from the pituitary and inhibition of ovarian cyclicity (1, 11). The suckling stimulus from the rat pups drives an increase in Prl release, which involves, at least in part, a suppression of tuberoinfundibular dopamine (TIDA) neural activity in the ARH (18, 24, 43). The lactating rat also displays a large increase in food and water intake to compensate for the fluid and energy demand of milk production. The mechanism leading to the increase in water intake is poorly understood; however, the increase in food intake is probably driven by changes in orexigenic and satiety factors, such as neuropeptide Y (NPY), agouti-related peptide, and proopiomelanocortin, in the ARH and dorsomedial hypothalamic nucleus (DMH) (2, 24, 26, 31). Lactation is also characterized by an overall reduction in the responsiveness to stress (9, 41), likely a result of altered CRH expression or activity in the PVH.

Because of the overlap between the centrally mediated role of ANG II to alter stress responses, reproductive hormone secretion, and Prl release and the dramatic changes in the HP axis during lactation, alterations in the brain RAS may be a mediating factor in one or more of these changes. One of the indicators of changes in sensitivity to any hormone is a change in the density of hormone binding sites. Therefore, the purpose of this study was to determine whether changes in the expression of brain ANG II receptor binding occur during lactation. Special attention was directed to ANG II binding in forebrain regions associated with the regulation of the HP axis.

	METHODS

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Animals and treatments. Pregnant (gestation day 18) and virgin cycling female rats (B&K Universal, Kent, WA) were received and housed individually on a 12:12-h light-dark cycle (lights on at 0700), with food and water available ad libitum. All animal procedures described in this paper were approved by the Oregon Regional Primate Research Center Institutional Animal Care and Use Committee. The day of delivery was considered day 0 postpartum. Litters were adjusted to eight pups on day 2 or 3 postpartum to allow for equivalent suckling stimuli and milk production. Vaginal swabs were taken daily and smears were examined microscopically to verify cyclicity in the virgin female rats. Each group contained 8-10 animals.

The rats were killed by decapitation on postpartum day 10 or 11 for the lactating rats and on diestrus for the cycling female rats. The brains were removed, frozen on crushed dry ice, and stored at 80°C until processed for in vitro receptor autoradiography. The brains were placed at 20°C 24-48 h before cryostat sectioning.

Receptor autoradiography. Rat brains were sectioned coronally in a cryostat at 20-µm thickness and thaw-mounted onto gelatin-coated slides. The sections were allowed to dry at room temperature and then stored at 80°C until the time of autoradiography.

Brain sections were thawed at ambient temperature (22-25°C) and preincubated in assay buffer (in mM: 150 NaCl, 1 EDTA, 0.1 bacitracin, and 50 sodium phosphate buffer at pH 7.2) for 30 min. Sections were then incubated in the same buffer containing 500 pM ¹²⁵I-labeled Sar¹,Ile⁸ ANG II (¹²⁵I-SI ANG II) for 2 h. The sections were rinsed by dipping them in two changes of distilled water, placing them for 1 min each into five changes of assay buffer, and again dipping in two changes of distilled water. The preincubation, incubation, and rinses were all carried out at ambient temperature (22-25°C). Immediately after rinsing, the sections were dried under a stream of cool air.

Nonspecific binding was determined by incubating brain sections in the ¹²⁵I-SI ANG II solution to which 3 µM ANG II had been added. Slides with radiolabeled tissue sections were mounted onto cardboard along with calibrated ¹²⁵I microscale standards (Amersham; Arlington Heights, IL), and apposed to X-ray film (Kodak, SB-5) for ~3 days. Films were developed in D-19 developer (Kodak) for 2 min at 20°C and fixed for 5 min in Kodak fixer.

Image analysis. The film images of ¹²⁵I binding to specific regions in the brain sections were evaluated for optical density to determine the amount of ANG II receptors in various brain regions. Films were evaluated using a Sony CCD video camera and an MCID M1 image analysis system (Imaging Research, St. Catherines, Ontario, Canada). Optical density readings were converted to units of fmol of ¹²⁵I-SI ANG II bound per gram tissue wet weight (37), calibrated with ¹²⁵I standards.

Images of brain sections were captured on a video monitor, and the desired nuclei were sampled for receptor density. The area sampled within each nucleus was measured for receptor density above an arbitrary computer-generated threshold level that enabled the irregular shape of the nucleus of interest to be completely sampled. Densitometric readings were made using a coding system to mask the group identity of the brains until the data were analyzed. At least two densitometric readings were obtained for each brain nucleus for each brain, and the average value was determined. The averages of the values for each nucleus were then included in the data set for statistical analysis. The size of the area sampled for each nucleus was also determined. If the average area of ANG II binding sampled for a nucleus in one group was >20% from the mean of the other group, brains whose nuclei had the highest or lowest sampling areas were resampled to adjust the area sampled to meet the arbitrary sample criterion. If there was damage to the brain section corresponding to the region of interest, values for that brain region were not included in the final data analysis. The density of ¹²⁵I-SI ANG II binding between the two groups was compared by Student's t-test. The level of significance was P < 0.05.

It should be noted that values reported are for a single, subsaturating concentration of ¹²⁵I-SI ANG II and do not represent maximal or even near-maximal binding density. Based on values reported by Rowe et al. (34), the average dissociation constant (K_D) value for ¹²⁵I-SI ANG II in predominantly AT₁ receptor-containing regions of the rat brain is 665 pM. In predominantly AT₂ receptor-containing regions, the average K_D is 2,409 pM. Thus at predominantly AT₁ receptor-containing brain regions, the ¹²⁵I-SI ANG II binding at 500 pM represents ~43% of maximal AT₁ receptor density. Where L = radioligand concentration: %maximal receptor density = L/(L + K_D). At predominantly AT₂ receptor-containing brain regions (34), the ¹²⁵I-SI ANG II binding reflects ~17% of maximal AT₂ receptor density.

Angiotensin receptor subtyping was not performed in this study due to the predominance of a single receptor subtype in most of the brain nuclei studied. In fact, nearly all of the brain regions studied, with the exception of the subthalamic nucleus (STN), contain almost exclusively AT₁ subtype receptor binding (4, 34) and mRNA (22). The STN expresses almost exclusively AT₂ receptor binding and mRNA (4, 22, 34).

	RESULTS

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Perihypothalamic nuclei. Six of the eleven perihypothalamic areas studied showed significant differences in ¹²⁵I-SI ANG II binding between lactation and diestrus (Table 1). There was a significant decrease in ¹²⁵I-SI ANG II binding in the SFO (27%), suprachiasmatic nucleus (29%; Fig. 1, C and D), ARH/ME (33%; Fig. 1, G and H), DMH (27%; Fig. 1, E and F), and lateral hypothalamic area (LHA; 23%; Fig. 1, E and F) during lactation compared with the diestrus. In contrast, there was a significant increase in ¹²⁵I-SI ANG II binding in the preoptic area (POA; 36%) in the lactating rat compared with the diestrous rat (Fig. 1, A and B). The area sampled as the POA consists of ANG II binding in many nuclei. However, binding appeared to increase in the whole region of the POA; therefore, it was sampled as a whole. The BSTV also showed a trend toward a decrease in ¹²⁵I-SI ANG II binding density (21%) in the lactating rat; however, the difference did not reach significance (P = 0.083). Other areas, including the ventral lateral diagonal band of Broca, median preoptic nucleus, and PVH, displayed no difference in binding density between the two groups.

View larger version (149K): [in this window] [in a new window]   Fig. 1.   Pseudocolor representation of ANG II binding during diestrus and lactation in female rat brain. Images shown represent 125I-labeled Sar1,Ile8 ANG II binding during diestrus (A, C, E, and G) and lactation (B, D, F, and H). Sampling areas were delineated by computerized image analysis system based on selection of an area outside of nucleus of interest to set an arbitrary threshold limit for sampling of nucleus of interest. A and B: rat brain sections at ~0.4 mm caudal to bregma. Double-headed arrow indicates ANG II binding in preoptic area. Short arrowhead indicates receptor binding in median preoptic nucleus. C and D: rat brain sections at ~0.8 mm caudal to bregma. Arrowhead indicates ANG II binding in lateral olfactory tract. White arrow indicates receptor binding in suprachiasmatic nucleus. E and F: rat brain sections at ~3.0 mm caudal to bregma. Arrowhead indicates ANG II binding in lateral hypothalamic area. Double-headed arrow indicates binding in dorsomedial hypothalamic nucleus. G and H: rat brain sections at ~3.6 mm caudal to bregma. Arrow indicates ANG II binding in arcuate nucleus. Arrowhead indicates receptor binding in median eminence. Pseudocolor standard bar (right) indicates density of ANG II binding (fmol/g) corresponding to colors in images. Area depicted in each panel is ~7.2 × 5.0 mm.

Other nuclei. The nucleus of the lateral olfactory tract (Fig. 1, C and D) and the STN also displayed significant decreases in ¹²⁵I-SI ANG II binding density in the lactating rat compared with the diestrous rat: 37% and 23%, respectively (Table 1). The piriform cortex (PC) displayed a near-significant decrease in binding density (30%, P = 0.059) in the lactating rat, whereas the ventral subiculum displayed no significant difference in binding density between the two groups.

	DISCUSSION

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Lactation in the rat is characterized by dramatic changes in the HP axis, as well as a more than doubling in food and water intake, compared with nonlactating rats (30). All of these physiological changes are necessary for the successful rearing of the pups. The RAS is a well-characterized regulator of several HP axis systems, as well as fluid and electrolyte balance. Therefore, changes in ANG II receptor expression in the hypothalamus during lactation could potentially explain some of the physiological changes that occur in this reproductive state.

Prl secretion is critical for milk production. In view of the demonstrated ability of brain ANG II to inhibit Prl secretion via stimulation of DA neuronal activity and release (20, 29) from the ARH and ME, respectively, successful milk production during lactation may entail a reduction in ANG II-mediated DA release from these regions. Therefore, the significant decrease in ANG II binding in ARH of the lactating rat (Table 1; Fig. 1, G and H) is expected and may be at least partially responsible for the decrease in TIDA neuronal activity during lactation (18, 24, 43). Although the ARH and ME were sampled together, it was evident that ANG II binding decreased in the lactating rat in both of these regions (Fig. 1, E-H). ANG II likely plays a role in the release of several hormones into the portal blood system (including GnRH, CRH, and/or DA) through activation of its receptors in the ME. In addition, ANG II receptor expression in the ARH and ME have been shown to be dependent on the presence of gonadal steroids, with ANG II receptors being highest when estrogen and progesterone are elevated (20, 35). The low levels of ANG II binding in these regions observed in this study are consistent with the low levels of estrogen during lactation.

Centrally administered ANG II has been shown to have a gonadal steroid-dependent action on LH release, with ANG II stimulating LH release in estrogen- and progesterone-treated ovariectomized (OVX) rats and inhibiting LH release in the untreated OVX rat (12, 38). Although the location of the ANG II receptors responsible for these actions on LH release is unknown, it is probable that ANG II receptors in brain regions containing estrogen and progesterone receptors, such as the POA, diagonal band of Broca, and BSTV (4, 16), play a role. The present data support this hypothesis, demonstrating a significant increase in ANG II receptor binding in the POA during lactation (Table 1; Fig. 1, A and B). Because the lactating rat is characterized as having nearly undetectable levels of estrogen, it is expected that ANG II would have an inhibitory effect on LH release. Therefore, the increase in ANG II binding in this region may be indicative of an increased inhibitory tone on GnRH neural activity. The stimulus driving the increase in ANG II receptors in this region remains unknown; however, pup suckling has been shown to stimulate expression of the immediate early gene c-Fos in this region (23), indicating an increase in neural activity (18). This area also contains a large population of GABA neurons, which may mediate the inhibitory actions of ANG II on GnRH neural activity.

Given the changes in the HP axis and the dramatic increase in water intake during lactation, it was hypothesized that there may be an increase in ANG II binding in the two major dipsogenic centers, the PVH and SFO. Surprisingly, there was no difference in ANG II binding in the PVH between lactation and diestrus (Table 1). In addition, the SFO actually displayed a significant decrease in ANG II binding during lactation (Table 1). However, because the level of ANG II is unknown during lactation, it is difficult to anticipate whether the lack of a change in ANG II binding in the PVH represents no change in angiotensinergic activity or whether the decrease in binding in the SFO actually represents a decrease in function. The potential change in brain ANG II levels will be discussed below. The reason for a lack of an increase in ANG II binding in these two dipsogenic control centers is unknown.

The role of ANG II in the DMH and LHA is not well defined; however, both brain regions are associated with regulation of food intake and body weight. The orexigenic peptide NPY is expressed in the DMH specifically during lactation, in a model of hyperphagia, and in specific genetic models of obesity (21, 24). The LHA is also a region that expresses numerous modulators of food intake, such as orexin, melanin-concentrating hormone, and CRH (see review, Ref. 10). Although ANG II is not classically associated with the regulation of food intake or body weight, two recent studies (6, 8) have demonstrated that chronic systemic ANG II administration caused a significant decrease in body weight. In addition, Brink et al. (6) reported that the decrease in body weight was directly associated with a decrease in food intake. Both the decreases in body weight and food intake occurred independently of the increase in blood pressure (6, 8). The mechanism by which systemic administration of ANG II causes a decrease in food intake is unknown. However, ANG II is a potent stimulator of CRH release, which is a known inhibitor of food intake (5, 32). In the present study, we demonstrate that ANG II receptor binding significantly decreases in the DMH and LHA during lactation (Table 1; Fig. 1, E and F), suggesting a possible decrease in angiotensinergic-associated hypophagia during lactation. The PC, which processes olfactory sensory information (36), also plays a role in the modulation of dietary amino acid intake, a function involving the noradrenergic system (14, 15). Interestingly, our group recently demonstrated that ANG II receptor binding in the PC was significantly reduced by lesioning of the medial forebrain bundle (17), the major brain stem noradrenergic projection to the forebrain. Therefore, the decline in ANG II binding in the PC of the lactating rat, although not reaching a significance of P < 0.05 (Table 1), may suggest a decrease in noradrenergic neurotransmission within this region, and subsequently a change in food intake. Further study is needed to investigate the potential role of ANG II in the regulation of food intake and the potential interactions between ANG II and orexigenic and satiety factors in these regions.

It is unknown what the endogenous levels of ANG II are in the plasma or brain of the lactating rat. Therefore, it is unknown whether the changes in ANG II receptor binding density are a result of homologous regulation caused by changes in ANG II levels or a direct result of the change in steroid environment. The lactating rat has elevated basal levels of glucocorticoids (42), which generally have a stimulatory effect on angiotensinogen (AON) mRNA expression in most regions of the brain (7, 33). However, there are some specific regions that display neither regulation by glucocorticoids nor even an inhibitory effect (7). The lactating rat is, therefore, likely to have region-specific changes in AON levels. Thus it is important to further investigate the levels of AON mRNA expression, as well as the enzymes responsible for the production of the active octapeptide, during lactation to determine the extent to which angiotensinergic function is modulated in this model.

Perspectives