Angiotensin II AT-1A receptor immunolabeling in rat medial nucleus tractus solitarius neurons: subcellular targeting and relationships with catecholamines.

The angiotensin II AT-1A receptor (AT-1A) is the major mediator of the hypertensive actions of angiotensin II (ANG II) in the medial nucleus of the solitary tract (mNTS). The localization of the AT-1A receptor at surface or intracellular sites is an important determinant of its signaling properties, including intercellular or intracrine communication. However, the spatial localization of this protein, particularly within small distal or intermediate size dendrites of mNTS neurons, is unknown. Within the mNTS, ANG II and catecholamines interact in the regulation of autonomic function; however, it is unknown if AT-1A receptors are present at functional sites in catecholamine containing dendrites, or are contacted by catecholamine containing axon terminals. We compared surface and intracellular distributions of the AT-1A receptor in dendritic processes from the mNTS using immunogold electron microscopy in conjunction with immunoperoxidase labeling for tyrosine hydroxylase (TH) and morphometric analysis. Collapsed across all AT-1A-labeled dendritic profiles, immunogold labeling was more frequent in intracellular sites as compared with the plasma membrane. Small (<0.6 microm) dendritic profiles contained a higher ratio of particles associated with the surface membrane when compared with larger profiles. Approximately 27% of all AT-1A receptor-labeled dendritic profiles also contained labeling for TH. Approximately 12% of dendritic profiles single labeled for the AT-1A receptor were contacted by TH containing axons or axon terminals. The present results provide the first quantitative demonstration of select plasmalemmal and intracellular localizations of AT-1A receptors in dendritic processes of mNTS neurons, including those containing TH, or contacted by catecholaminergic axon terminals. These results suggest that AT-1A receptors are positioned for modulation of catecholamine signaling in the mNTS.

Angiotensin type 1a receptors on corticotropin-releasing factor neurons contribute to the expression of conditioned fear.

Although generally associated with cardiovascular regulation, angiotensin II receptor type 1a (AT1a R) blockade in mouse models and humans has also been associated with enhanced fear extinction and decreased post-traumatic stress disorder (PTSD) symptom severity, respectively. The mechanisms mediating these effects remain unknown, but may involve alterations in the activities of corticotropin-releasing factor (CRF)-expressing cells, which are known to be involved in fear regulation. To test the hypothesis that AT1a R signaling in CRFergic neurons is involved in conditioned fear expression, we generated and characterized a conditional knockout mouse strain with a deletion of the AT1a R gene from its CRF-releasing cells (CRF-AT1a R((-/-)) ). These mice exhibit normal baseline heart rate, blood pressure, anxiety and locomotion, and freeze at normal levels during acquisition of auditory fear conditioning. However, CRF-AT1a R((-/-)) mice exhibit less freezing than wild-type mice during tests of conditioned fear expression-an effect that may be caused by a decrease in the consolidation of fear memory. These results suggest that central AT1a R activity in CRF-expressing cells plays a role in the expression of conditioned fear, and identify CRFergic cells as a population on which AT1 R antagonists may act to modulate fear extinction.

Rat epididymis contains functional angiotensin II receptors.

Specific angiotensin II (Ang II) binding and functional responses have been studied in a number of male and female reproductive structures, but to date have not been characterized in the epididymis. Some epididymal functions, including smooth muscle contraction and regulation of fluid and electrolyte balance, are mediated by Ang II in other tissues. This study demonstrates specific, saturable (63 fmol/mg protein), high affinity (Kd, less than 1 nM) Ang II-binding sites in the epididymis. These binding sites, localized in the circumference of the epididymal tubule and most concentrated within the proximal cauda, are present throughout the caput, corpus, and remaining cauda epididymis. Ang II caused powerful expulsions of spermatozoa in segments of epididymis in situ. These results suggest Ang II involvement in epididymal function.

Variations in angiotensin-II release from the rat brain during the estrous cycle.

To investigate the hypothesis that the release of angiotensin-II (AII) in the rat brain increases on the day of proestrus, samples of cerebrospinal fluid (CSF) and interstitial fluid from the general region of the bed nucleus of the stria terminalis pars ventralis in the preoptic-anterior hypothalamic area were monitored for AII-immunoreactive material (AII-ir) using push-pull cannulas. Samples of CSF were obtained on the day of proestrus and diestrus day 1 at 30-min intervals from 1200-1600 h. Samples of interstitial fluid were obtained at 25-min intervals from 0930-1600 h. The rate of release of AII-ir into CSF was significantly greater on proestrus compared to diestrus day 1, and in the early afternoon of proestrus compared to the late afternoon. In five of seven rats and in the overall comparison of AII-ir release from the preoptic-anterior hypothalamic area, significantly more AII-ir was released on the day of proestrus vs. diestrus day 1. These observations are consistent with previous studies suggesting that brain AII may play a role in the regulation of LH release on the day of proestrus.

Vasopressin V2 receptor binding is down-regulated during renal escape from vasopressin-induced antidiuresis.

This study evaluated whether renal escape from vasopressin-induced antidiuresis is associated with alterations of vasopressin V2 receptor binding in the kidney inner medulla. A radioligand binding assay was developed using a novel iodinated vasopressin V2 receptor antagonist to analyze vasopressin V2 receptor binding in kidney inner medullary tissue from three groups of rats: normal rats maintained on ad libitum water intake, rats treated with 1-deamino-[8-D-arginine]vasopressin (DDAVP), and rats treated with DDAVP that were also water loaded to induce renal escape from antidiuresis. Analysis of the binding data showed that DDAVP treatment reduced vasopressin V2 receptor binding to 72% of normal levels. Water loading induced a marked further down-regulation of vasopressin V2 receptor binding. This receptor down-regulation began by day 2 of water loading, which correlated with the initiation of renal vasopressin escape; by day 3 of water loading, vasopressin V2 receptor expression fell to 43% of DDAVP-treated levels. No differences in vasopressin V2 receptor binding affinities were found among the three groups. This study demonstrates that vasopressin V2 receptor binding capacity is down-regulated during renal escape from vasopressin-induced antidiuresis and suggests that both vasopressin-dependent mechanisms as well as vasopressin-independent mechanisms associated with water loading are involved in this receptor down-regulation.

Rat (Ile5) but not bovine (Val5) angiotensin raises plasma norepinephrine in rats.

A part of the vasoconstrictor activity of angiotensin II (AII) may result from its ability to enhance norepinephrine (NE) release from sympathetic noradrenergic nerve terminals. To investigate this proposed pressor mechanism of AII, the effects of intravenous (i.v.) infusion of AII on blood pressure and plasma catecholamines in pithed rats were determined. Two naturally occurring angiotensins, valine5 AII (bovine) and isoleucine5 AII (rat), were administered in equal (72 ng/min) doses. Valine5 AII caused an 80% increase in mean arterial pressure (MAP) from 54 +/- 4 to 97 +/- 19 mm Hg. Isoleucine5 AII caused an 82% increase in MAP from 49 +/- 5 to 89 +/- 18 mm Hg. Neither angiotensin caused a change in heart rate, suggesting that pithing completely destroyed the central baroreceptor reflex mechanism. Plasma catecholamines were differentially affected by the peptides:isoleucine5 AII significantly increased plasma NE concentration by 82% compared to saline-infused rats (p less than 0.01). Valine5 AII did not significantly affect plasma NE concentration. Plasma dopamine and epinephrine concentrations were not significantly altered by infusion of either analog. Despite the significant increases in plasma NE concentrations with isoleucine5 in AII-infusion rats, there was no correlation between plateau MAP or the percent increase in MAP and plasma NE concentrations of individual animals within this group. The ability of angiotensin to elevate MAP, increase NE release from sympathetic nerve terminals, as well as potential differences in the actions of angiotensins in different species, and angiotensin receptor heterogeneity, are discussed.

Basal levels of plasma epinephrine and norepinephrine in the dog.

Conscious (n = 62) and anesthetized (n = 34) dogs were studied to establish basal levels and ranges for plasma epinephrine (E) and norepinephrine (NE) in this species. Trained conscious dogs were familiarized to recording conditions and personnel for 2 to 3 weeks and acclimatized to the laboratory for at least 15 minutes prior to blood sampling from a chronically implanted catheter. Their basal values were 65 +/- 47 pg/ml for E and 145 +/- 58 pg/ml for NE, which were significantly lower (p less than 0.05) than values in a second group of conscious dogs trained in the same manner but sampled soon after arrival to the laboratory (E = 144 +/- 93 pg/ml; NE = 193 +/- 86 pg/ml). Catecholamine levels in dogs anesthetized with one of three different regimes commonly used in cardiovascular studies were shown to be similar to the basal values found in conscious dogs acclimatized to the laboratory. The weak correlations found between basal plasma catecholamines and hemodynamic variables in all groups of conscious dogs reflect the complexity of factors interacting with the sympathetic nervous system in the maintenance of arterial pressure. These results document the variability that can be expected when using catecholamine levels as an index of sympathetic nervous system activity and the necessity of standardizing conditions for sample collection.

Catecholamines and serotonin in the area postrema of normal and sodium-depleted dogs.

Sodium depletion, a maneuver that is accompanied by a 14-fold elevation of plasma renin activity (PRA), alters the norepinephrine concentration of the canine area postrema (AP), a circumventricular organ of the 4th ventricle known to be sensitive to circulating angiotensin II. The norepinephrine concentration of the AP after 3 weeks of sodium depletion decreased by 43%, whereas the concentration of epinephrine and dopamine and the activity of phenylethanolamine-N-methyltransferase (PNMT) did not change. In the pyramidal tract (PT) and choroid plexus (CP) catecholamines were present in significantly lower amounts than in the AP; their concentrations were unaffected by sodium depletion in the PT, but in the CP the norepinephrine concentration was reduced. Serotonin was present in the AP but its concentration was unaltered by sodium depletion. These findings provide evidence that sodium depletion produced an alteration in the concentration of norepinephrine of the area postrema without any change in the concentration of epinephrine, dopamine or serotonin.

Alterations in plasma and cerebrospinal fluid norepinephrine and angiotensin II during the development of renal hypertension in conscious dogs.

The levels of norepinephrine (NE), epinephrine (E), and angiotensin II immunoreactivity (AIIir) in plasma and in cerebrospinal fluid (CSF) were measured in eight conscious dogs before and during a 28-day period in the development of two-kidney, one clip (2K1C) hypertension produced by a two-step procedure. The early phase (less than 7 days) of hypertension following partial constriction of the renal artery was accompanied by tachycardia and increases in concentrations of NE and AIIir in both plasma and CSF; E did not change. One week later blood pressure remained elevated (107 +/- 2 after vs 88 +/- 2 mm Hg before clipping, p less than 0.05), but other variables returned to control values. Occlusion of the partially constricted renal artery caused severe hypertension that was initially associated with a transient decrease in levels of NE in both plasma and CSF and a sustained rise in plasma and CSF concentrations of AIIir that persisted for as long as 2 weeks after the second operation. None of these effects was seen in nine sham-operated dogs. Since activation of the renal pressor system is associated with time-related changes in the concentrations of NE and AII in both plasma and CSF, these observations indicate early involvement of both sympathetic and renin-angiotensin systems in the pathogenesis of renovascular hypertension.

Regulation of adrenal angiotensin receptor subtypes: a possible mechanism for sympathectomy-induced adrenal hypertrophy.

OBJECTIVE: Previous studies indicate that the adrenal gland plays a compensatory role in the maintenance of blood pressure in chemically sympathectomized rats. However, the mechanisms responsible for compensatory adrenal responses are poorly understood. This study examined the regulation of adrenal growth and type 1 A, 1 B, and type 2 angiotensin II (Ang II) receptor (AT1A, AT1B and AT2) expression in the adrenal gland induced by sympathectomy. METHODS: Five-week-old male Sprague-Dawley rats were treated with either guanethidine (50 mg/kg per day, intraperitoneally) or vehicle for 5 weeks. Norepinephrine and epinephrine levels in the atrium of the heart were measured by high-pressure liquid chromatography. Plasma renin activity was determined by radioimmunoassay. Adrenal AT1 and AT2 receptor density was determined by radioligand binding assay. Adrenal AT1A, AT1B and AT2 mRNA levels were determined by Northern blot analysis. RESULTS: Norepinephrine and epinephrine levels in the atrium of the heart were decreased 86% (P < 0.0001) and 58% (P < 0.05) by guanethidine treatment, respectively. Plasma renin activity was decreased 71% (P< 0.001) in guanethidine-treated rats compared with vehicle. In contrast, the ratio of adrenal to body weight was increased 38% in guanethidine-treated rats compared with vehicle (P< 0.001). Adrenal AT1 and AT2 receptor density was increased by guanethidine treatment (P< 0.05). Adrenal mRNA levels for AT2 (P< 0.001) and AT1A (P< 0.01), but not AT1B (P>0.05), were increased in guanethidine-treated rats compared with vehicle (P< 0.01). There were positive correlations between adrenal weight and AT2 (r = 0.9, P< 0.001) and AT1A (r = 0.6, P< 0.05) but not AT1B (r = - 0.01, P > 0.05) expression. CONCLUSIONS: Impairment of the sympathetic nervous system with guanethidine withdraws the normal stimulation of this system on the circulating renin-angiotensin system, but upregulates the expression of adrenal Ang II receptors. Increased expression of adrenal AT2 and AT1A receptors may play an important role in adaptive adrenal hypertrophy and hormonal responses to sympathectomy.

Low-calcium diet increases blood pressure and alters peripheral but not central angiotensin II binding sites in rats.

The mechanism by which low-calcium (Ca) diet causes hypertension is unknown. We investigated angiotensin II (Ang II) receptor binding in brain, adrenals and urinary bladders in male Sprague-Dawley rats pair-fed a low-Ca (0.005% Ca; 0.5% P) and normal-Ca (1.4% Ca) diet for 8 weeks beginning at 4 weeks of age. The Ang II receptor sites in hypothalamus-thalamus-septum (HTS), adrenal glands and urinary bladder smooth muscle were measured by saturation isotherm binding using 125I-sarcosine1isoleucine8 Ang II (125I-SI Ang II). Systolic blood pressure was determined at 2-week intervals by tail-cuff method. Serum total Ca, Na+, K+ aldosterone and Ang II and bone density and mineral content were determined at the time of sacrifice. Chronic Ca deficiency in rats raised blood pressure and decreased Ang II receptor density in bladder smooth muscles and tended to increase adrenal Ang II receptors. Serum Ca. bone density and mineral content were significantly lower in the Ca-deficient rats, while serum Na+ was elevated in this group. Serum Ang II and aldosterone were unaltered after the 8-week dietary regimen. Possible mechanisms for the hypertensive actions of reduced dietary Ca intake involving the renin-angiotensin-aldosterone system are discussed.

Topographic probes of angiotensin and receptor: potent angiotensin II agonist containing diphenylalanine and long-acting antagonists containing biphenylalanine and 2-indan amino acid in position 8.

A series of phenylalanine-mimicking amino acids with increasing conformational restraint were prepared and incorporated into angiotensin II, in order to develop topographic probes of angiotensin useful for probing receptor boundaries by molecular graphics analysis and for conformational analysis of the ligand by NMR. In binding studies, all analogues displayed high affinity for rat uterus (Ki of 0.74-6.08 nM) and brain (0.46-1.82 nM) receptors. In smooth muscle (rat uterus) contraction assay, the diphenylalanine-containing [Sar1,Dip8]AII and [Sar1,D-Dip8]AII were potent agonists with respectively 284% and 48% activity of [Asn1]AII. In contrast, the biphenylalanine-containing [Sar1,Bip8]AII, [Sar1,D-Bip8]AII, and the 2-indan amino acid containing [Sar1,2-Ind8]AII were potent inhibitors, approximately 9, 2, and 1.4 times more effective than a standard antagonist, [Sar1,Leu8]AII. Their respective pA10 values in rat uterus assay were 8.87, 8.70, and 8.82. By comparison, the pA10 value for [Sar1,Leu8]AII was 8.35. In rats, a single dose of 10 micrograms of [Sar1,2-Ind8]AII or [Sar1,Bip8]AII produced prolonged blockade of the pressor response toward angiotensin II for over 90 min. The very different pharmacological profiles of these rigid aromatic analogues suggest that the angiotensin receptor activation site consists of a relatively wide and elongated pocket with a narrow opening.

Distribution of [125I]angiotensin II binding sites in the rat brain: a quantitative autoradiographic study.

Angiotensin II receptors have been localized by quantitative autoradiography in the rat central nervous system after labeling with [125I]angiotensin II. A highly discrete distribution of these receptors was found throughout the rat brain. The highest density was seen in regions of the medulla, hypothalamus and circumventricular organs where angiotensin II could potentially produce cardiovascular, dipsogenic and neuroendocrine responses. The distribution of angiotensin II receptors correlates relatively well with the previously reported distribution of angiotensin immunoreactive nerve terminals as well as areas determined by various physiological techniques to be sensitive to angiotensin II. Finally, the anatomical localization of angiotensin II receptor populations has revealed several areas of the brain where the effects of this peptide have not been investigated. Many of these nuclei are involved in the transmission and processing of somatic and visceral sensory information. These results suggest a broader role for the central renin-angiotensin system in modulating several types of sensory input.

Angiotensin II subtype 1A (AT1A) receptors in the rat sensory vagal complex: subcellular localization and association with endogenous angiotensin.

Angiotensin II (Ang II) type 1 (AT1) receptors are prevalent in the sensory vagal complex including the nucleus tractus solitarii (NTS) and area postrema, each of which has been implicated in the central cardiovascular effects produced by Ang II. In rodents, these actions prominently involve the AT1A receptor. Thus, we examined the electron microscopic dual immunolabeling of antisera recognizing the AT1A receptor and Ang II to determine interactive sites in the sensory vagal complex of rat brain. In both the area postrema and adjacent dorsomedial NTS, many somatodendritic profiles were dually labeled for the AT1A receptor and Ang II. In these profiles, AT1A receptor-immunoreactivity was often seen in the cytoplasm beneath labeled portions of the plasma membrane and in endosome-like granules as well as Golgi lamellae and outer nuclear membranes. In addition, AT1A receptor labeling was detected on the plasma membrane and in association with cytoplasmic membranes in many small axons and axon terminals. These terminals were morphologically heterogeneous containing multiple types of vesicles and forming either inhibitory- or excitatory-type synapses. In the area postrema, AT1A receptor labeling also was detected in many non-neuronal cells including glia, capillary endothelial cells and perivascular fibroblasts that were less prevalent in the NTS. We conclude that in the rat sensory vagal complex, AT1A receptors are strategically positioned for involvement in modulation of the postsynaptic excitability and intracrine hormone-like effects of Ang II. In addition, these receptors have distributions consistent with diverse roles in regulation of transmitter release, regional blood flow and/or vascular permeability.

Pituitary lactotrope expresses transforming growth factor beta (TGF beta) type II receptor mRNA and protein and contains 125I-TGF beta 1 binding sites.

Transforming growth factor beta 1 (TGF beta 1) has recently been shown to be produced in the prolactin (PRL)-secreting lactotropes of the pituitary gland. TGF beta 1 inhibits lactotropic secretion and proliferation, and the production of TGF beta 1 in lactotropes is reduced during lactotropic growth following estrogen treatment in ovariectomized rats. In many estrogen-responsive tissues, TGF beta 1 has been shown to exert its effect by binding to TGF beta 1 type II receptors (T beta R II) at the cell surface. In this study, we sought to ascertain whether T beta R II is involved in TGF beta 1 action on lactotropes by determining the changes of T beta R II mRNA and protein levels and specific 125I-TGF beta 1 binding sites on the lactotropes during estrogen-induced proliferation of lactotropes in Fischer 344 rats. Double immunohistochemical procedures were employed to identify immunoreactive T beta R II in PRL-reactive cells. The majority of T beta R II-reactive cells in the anterior pituitary were observed to be lactotropes. Dual immunohistochemistry and in situ hybridization procedures also indicated that lactotropes were the major cell types containing T beta R II mRNA hybrids. Both the levels of immunoreactive T beta R II protein and in situ T beta R II mRNA hybrids in the pituitary were significantly decreased in ovariectomized rats after 15 days of estrogen treatment. Determination of 125I-TGF beta 1 binding sites in lactotropes by double immunohistochemistry and receptor autoradiography also revealed specific binding sites of 125I-TGF beta 1 in lactotropes in the anterior pituitary. 125I-TGF beta 1 binding in the anterior pituitary was also reduced following estrogen treatment in ovariectomized rats. These data suggest that down-regulation of T beta R II may be an important mechanism of estrogen action on lactotropic cell growth and PRL secretion, and further support the notion that TGF beta 1 controls lactotropic function by autocrine/paracrine mechanisms.

Angiotensin II and non-angiotensin II displaceable binding sites for [3H]losartan in the rat liver.

By virtue of the more than 1000-fold selectivity of losartan (DuP 753) for the AT1 angiotensin II (AII) receptor subtype compared with the AT2 subtype, [3H]losartan may be a useful radioligand for studies of the AT1 receptor subtype. Comparison of Bmax values in the liver obtained from saturation isotherms using [3H]losartan (Bmax = 194 pmol/g tissue) and [125I]sarcosine1,isoleucine8 angiotensin II (Bmax = 20 pmol/g tissue) indicated that the AII receptor concentration was approximately 10% that of the [3H]losartan binding sites. In addition, AII at concentrations as high as 10 microM displaced less than one-third of specific [3H]losartan binding in the liver and less than 80% in the whole adrenal. The presence of non-AII displaceable [3H]losartan binding in the liver did not appear to result from metabolism of the radioligand since HPLC analysis of free and bound 3H revealed that greater than 90% of the 3H eluted at the same time as the parent [3H]losartan. This suggests that [3H]losartan binds with high affinity to a site(s) other than angiotensin II receptors in the rat liver.

Effects of peptidase inhibitors on binding at angiotensin receptor subtypes in the rat brain.

Sulfhydryl reducing agents affect angiotensin II (AII) receptor binding differentially at AT1 and AT2 sites. Consequently, sulfhydryl reducing agents are now used infrequently in AII receptor binding assays. In this regard, the present autoradiographic study evaluates the effects of additional peptidase inhibitors on AII receptor binding and radioligand integrity. EDTA at 5 mM enhanced binding similarly, by about 70%, at both AT1 and AT2 binding sites, whereas bacitracin (10(-4) M) did not affect binding at either site. In contrast, addition of phenanthroline and bovine serum albumin (BSA) increased binding at AT1 sites 2.3-fold, whereas binding at AT2 sites was affected minimally. Degradation of 125I-[Sar1,Ile8]-AII (125I-SIAII) was determined by HPLC analysis of samples before and after incubation with tissue in each buffer. Omission of bacitracin from buffers reduced the recovery of intact radioligand to 83-87%, while recovery exceeded 94% in the presence or absence of all other buffer constituents. These results suggest that degradation of 125I-SIAII is minimal in large volume in vitro receptor autoradiography studies of rat brain AII receptors. Further, the beneficial effects on radioligand binding caused by buffer constituents such as EDTA, phenanthroline, and BSA were not due to their ability to protect the radioligand from enzymatic degradation. Because these constituents (and possibly others) had differential effects on binding with respect to receptor subtypes, caution should be used when interpreting or comparing binding data obtained from various laboratories utilizing different buffer components.

Angiotensin II and the locus coeruleus.

The locus coeruleus (LC) is a putative site of action for angiotensin II in the brain. Immunocytochemical studies have identified angiotensin II-like immunoreactive material in nerve terminals innervating the LC, and the LC contains one of the highest densities of angiotensin II receptor binding sites in the rat brain. Recent studies using selective neurotoxins suggest that the binding sites for angiotensin II in the LC are present on noradrenergic perikarya. Angiotensin II receptors are now known to exist as two subtypes that are distinguishable both pharmacologically and biochemically. Radioligand binding studies using agonists and antagonists selective for these angiotensin II receptor subtypes indicate that the rat LC contains a mixture of the two known angiotensin II receptor subtypes, but that the PD123177-sensitive AII beta receptor subtype is predominant. Comparisons of spontaneously hypertensive rats with normotensive rats indicates that angiotensin II and its receptors in the LC are elevated in the hypertensive rat strain. Studies of the biochemical and physiological actions of angiotensin II in the LC have not yet established an agreed-upon function for angiotensin II in this nucleus.

Dietary NaC1 during pregnancy and lactation: effect on brain angiotensin II receptors and behavior.

Female rats were fed diets containing either a basal (0.12%), mid- (1%) or high (3%) level of NaCl during pregnancy and lactation. Plasma aldosterone was elevated approximately 5- and 15-fold in dams fed basal compared with either the mid- or high-NaCl diets at the end of both pregnancy and lactation (Postnatal Day 21), respectively. Dams fed basal diet and killed at the end of lactation had a higher density of angiotensin II receptors in the organum vasculosum laminae terminalis, paraventricular hypothalamus, and median preoptic nucleus than did rats fed either mid- or high-NaCl diets. Other dams, treated identically, were returned to rodent chow (approximately 0.2% NaCl) at the end of lactation for intake tests during the next week. Dams that had received basal diet did not differ from mid-NaCl and high-NaCl groups in sodium appetite induced by either acute sodium depletion or mineralocorticoid administration but showed the lowest spontaneous intake of NaCl solution.