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Angiotensin II AT1 receptors are highly localized in the dorsomedial arcuate nucleus. AT1 receptor number is very low during proestrus and in ovariectomized and male rats, and is high only during the estrus phase of the estrous cycle and after ovariectomized rats receive a sequential estrogen-progesterone treatment. Our results suggest that the mechanism of the estrogen-progesterone inhibition of the prolactin surge may involve the selective stimulation of dorsomedial arcuate AT1 receptors.

High-affinity melatonin receptors are expressed in the tail artery of the rat and arteries forming the circle of Willis of the rat and certain primates. The characteristics of the vascular melatonin receptors seem to be similar to the ones described in the central nervous system. The expression of vascular melatonin receptors in the rat is differentially regulated by factors such as strain and age, and in the female by reproductive hormones. Functional studies using the caudal artery of the rat suggest that melatonin regulates vascular tone. The highly restricted distribution of melatonin receptors in arteries involved in regulating blood flow to areas involved in heat dissipation suggests that vascular melatonin receptors may have a specific function in thermoregulatory homeostasis.

Angiotensin II receptors of the AT1 subtype were very highly expressed in medroxyprogesterone-induced ductal adenocarcinomas of the mammary gland in BALB/c mice. AT1 receptors are associated only to neoplastic epithelial cells. Lobular adenocarcinomas expressed very few AT1 receptors and expressed AT2 receptors only in areas corresponding to peritumoral connective tissue. Binding to angiotensin converting enzyme was present in all adenocarcinomas studied and was higher in ductal than in lobular adenocarcinomas. Normal mammary gland did not express either angiotensin II receptors or angiotensin converting enzyme. The present results are the first demonstration of angiotensin receptor subtypes and converting enzyme in mammary adenocarcinomas differentially expressed in tumors of ductal and lobular origin. Localization of receptor subtypes could be useful to study the differentiation of mammary cells during experimental mammary carcinogenesis in mice.

We investigated the effect of angiotensin AT1 and AT2 receptor blockade on the upper limit of CBF autoregulation in pentobarbital-anesthetized rats. CBF was measured by laser-Doppler flowmetry from the parietal cortex and MABP was increased by intravenous phenylephrine infusion. Neither the AT1 antagonist losartan nor the AT2 ligand PD 123319 nor angiotensin II (ANG II) in the presence of losartan affected baseline CBF. When the blood pressure was increased in the control group, CBF remained fairly constant up to 145 mm Hg and increased steeply after 150 mm Hg. Both PD 123319(7–10 mg/kg) and losartan (1–10mg/kg) shifted the upper limit of CBF autoregulation toward higher pressures. Intravenous infusion of PD 123319 was more effective than bolus injection. The losartan effect was dose dependent. Selective stimulation of AT2 receptors with an intravenous ANG II infusion (0.54 μg/min) in the presence of losartan did not reverse the effect of losartan on CBF autoregulation, but, on the contrary, appeared to further shift the upper limit of autoregulation toward higher pressures. The results implicate a role for both AT1 and AT2 angiotensin receptors in the regulation of CBF.

Insulin-like growth factor I (IGF-I) and the closely related IGF-II are homologous to proinsulin and share many of insulin’s effects on cell metabolism. In addition they stimulate cellular differentiation and proliferation1. Circulating IGF-I originating mainly from the liver, has classical endocrine actions2, however, local IGF-I production in many other tissues suggests an autocrine/paracrine role as we113. Most biological actions of the IGFs are mediated through the IGF-I receptor (IGFR-I) which has intrinsic tyrosine kinase activity4,5 Specific, high-affinity, insulin-like growth factor binding proteins (IGFBPs), present in the plasma and extracellular space, also bind the IGFs, modulating their interaction with the IGF-I receptor and perhaps targeting IGFs to specific loci6. To date, six different IGFBPs have been characterized7, among which IGFBP2 is particularly abundant in the pituitary8.

RECENTLY two subtypes of angiotensin receptors have been described, AT1 and AT2. Currently used radiolabeled agonists and antagonists are not able to discriminate between these receptor subtypes. Here we characterize the use of [125I] CGP 42112A, a novel, specific ligand for AT2 receptors, in a membrane binding assay and in autora-diography of brain sections of 2 week old rats. [125I] CGP 42112A bound with high affinity and autoradiography revealed binding selectively localized to areas known to express the AT2 receptor subtype only. CGP 42112A, angiotensin II, angiotensin III and PD 123177 competed for [125I] CGP 42112A binding, with potencies consistent with high affinity and specific binding to AT2 receptors. Thus [125I] CGP 42112A will be a useful new tool to study AT2 receptors.

Quantitative autoradiography was used to compare melatonin receptors in brain areas and arteries of young (4 weeks old) and adult (14 weeks old) spontaneously hypertensive rats (SHR) to those in age-matched normotensive controls, Wistar-Kyoto (WKY) rats. Age and strain influenced the number of melatonin receptors in an anatomically selective manner, and the most striking changes occurred in arterial receptors. Melatonin receptors were not detectable in the anterior cerebral arteries of adult SHR. In the caudal artery, melatonin receptors decreased with age in both strains, but the decrease was more pronounced in SHR. When compared to age-matched WKY rats, the number of caudal artery receptors was higher in young and lower in adult SHR. The number of melatonin receptors was higher in the area postrema of adult SHR when compared to adult WKY rats, but in the suprachiasmatic nucleus, no such differences between the two strains were present. Alterations in receptor density were not accompanied by changes in binding affinity. Our results indicate that in the rat melatonin receptors show different developmental patterns according to location and that the receptors may be expressed differentially in genetic hypertension.

LARGE cerebral arteries have been reported to contain angiotensin receptors that are exclusively of the AT2 subtype. We measured the effect of the AT2 receptor selective ligand PD 123319 on cerebral blood flow (CBF) in rats, using laser-doppler flowmetry. PD 123319 (1-10 mg kg-1) dose-dependently inhibited the increase in CBF, when the blood pressure was increased by a norepinephrine infusion. However, PD 123319 did not alter baseline CBF at normal blood pressures. Therefore PD 123319 appears to interfere with the autoregulatory mechanisms of CBF. The participation of AT2 receptors in the regulation of CBF confirms a physiological role for this receptor subtype, and may give clues for future treatment of various cerebrovascular disorders.

We localized and characterized angiotensin II AT1 and AT2 receptors in the skin of 2-week-old rats during experimental wound healing. Both AT1 and AT2 were present in the skin. Three days after wounding, the expression of angiotensin II receptors was significantly enhanced in the dermis as well as in a localized band within the superficial dermis of the skin surrounding the wound. The major proportion of this increase was due to angiotensin II AT2 receptors. Our results suggest a physiological role for AT2 receptors in the process of tissue repair.

I. Introduction Angiotensin(ANG II) was discovered about 50 yr ago (1, 2), and a complete peripheral system for the formation and metabolism of ANG II was soon characterized. It was named the renin-angiotensin system, based on the rate-limiting enzyme, renin, and the active principle, ANG II (3). In the classical peripheral RAS system, a precursor molecule, angiotensinogen (AOGEN), originates in the liver and is cleaved to the essentially inactive decapeptide angiotensin I (ANG I) by an aspartyl protease, renin, synthesized and released to the circulation mainly by the juxtaglomerular cells of the kidney. ANG I is converted to ANG II, the effector peptide of this system, by the angiotensin-converting enzyme (ACE) (4). Circulating ANG II acts on high affinity ANG II receptors located in the vascular smooth muscle, zona glomerulosa of the adrenal gland, and kidney to produce vasoconstriction, aldosterone release, and sodium retention, respectively. Thus, the peripheral RAS is a hormonal system with importan...

Quantitative autoradiography was used to determine the subtype of ANG receptors in the red pulp of the rat spleen. The AT1 antagonist DuP 753 competed for ANG binding with high affinity; binding was abolished by dithiothreitol. The AT2 competitor CGP 42112 A showed lower affinity, and the AT2 competitor PD 123177 did not affect binding at 10(-5) M. These data indicated the presence of only AT1 receptors. AT1 receptor number was similar in immature (2 weeks old) and adult (8 weeks old) rats. Binding was sensitive to guanine nucleotides, suggesting an association with G-proteins. Angiotensin II, at a dose of 10(-7) M, stimulated inositol phosphate formation 33% over control values in spleen from 8-week-old rats. This effect was significantly blocked by 10(-5) M DuP 753. We suggest a possible role of AT1 receptors in the regulation of splenic volume, blood flow, and lymphocyte function.

Angiotensin II receptor number was higher in superior cervical ganglia of 2-week-old when compared to 8-week-old rats. In both young and adult rats, specific binding of [125I][Sar1]angiotensin II was displaced competitively by the AT1-receptor antagonist DuP 753 but not by the AT2-receptor competitor PD 123177. In ganglia from adult rats, DuP 753 competed with an IC50 of 113 nM. The stable guanine nucleotide GTP gamma S inhibited binding of [125I][Sar1]angiotensin II in young and adult rats by approximately 50% with IC50 values of 105 and 120 nM, respectively, suggesting that the angiotensin receptor is G-protein linked. Angiotensin II at a dose of 1 microM stimulated inositol phosphate formation 58% over control values in superior cervical ganglia from 8-week-old rats. This effect was totally blocked by 10 microM DuP 753 but not by 10 microM PD 123177. Our findings demonstrate that rat superior cervical ganglia contain AT1-type angiotensin receptors that are probably G-protein linked, and their stimulation results in increased inositol phospholipid metabolism.

We used quantitative autoradiography to characterize angiotensin-II (ANG-II) receptor subtypes in areas of the rat basal forebrain involved in pituitary control. ANG binding was totally displaced by the selective AT1 receptor subtype antagonist DuP 753 and was inhibited by dithiothreitol in median eminence, infundibular stem, paraventricular nucleus, subfornical organ, median preoptic nucleus, anterior pituitary, and the forebrain ANG-II receptor band, which extends from the subfornical organ to the vascular organ of the lamina terminalis, including the lamina terminalis and a continuous band of receptors reaching the paraventricular nucleus. In these areas, binding was not affected by the selective AT2 competitors PD 123177 or CGP 42112 A, indicating the presence of AT1 and the absence of AT2 receptors. AT1 binding was higher in the external layer and lateral parts of the median eminence. Conversely, ANG binding in the dura mater encapsulating the pituitary, in the vessels adjacent to this part of the dura mater, presumably branches of the hypophyseal arteries, and in anterior cerebral arteries was displaced by PD 123177 and CGP 42112 A, and was unaffected by dithiothreitol or DuP 753, indicating the presence of AT2 receptors in these structures. Our results implicate AT1 receptors in the central neural regulation of pituitary function. AT2 receptors may play a role in the regulation of blood flow to the basal forebrain and the pituitary gland.

Quantitative autodradiography was used to localize and characterize atrial natriuretic peptide (ANP) receptors in the rat brain and to study their regulation. Peptide receptors are selectively located to circumventricular organs outside the blood brain barrier, such as the subfornical organ, and to brain areas involved in fluid and cardiovascular regulation. Dehydration, either by water deprivation of normal rats, or chronic dehydration present in homozygous Brattleboro rats lacking vasopressin, results in large increases in ANP binding in receptor number in the subfornical organ. In the deoxycorticosterone acetate (DOCA)–salt hypertensive model, only salt treatment, but not DOCA alone or the combination of DOCA–salt, increased the ANP receptor number in the subfornical organ and the choroid plexus. Both young and adult genetically hypertensive rats have a greatly decreased ANP receptor number in the subfornical organ and the choroid plexus. Selective displacement with an inactive analog lacking the disulfide bond (ANP 111–126) suggests that genetically hypertensive rats may lack C (clearance) atrial natriuretic peptide receptors. Our results implicate brain atrial natriuretic peptide receptors in the central response to alterations in fluid regulation and blood pressure.Key words: quantitative autoradiography, fluid regulation, atriopeptins, genetic hypertension, atrial natriuretic peptide binding sites.

Summary Quantitative autoradiography was used to characterize angiotensin AT1 and AT2 receptors, in the rat aorta at three developmental ages; embryonic day 18 (E18), and postnatal weeks 2 and 8. The expression of angiotensin receptors was higher in the aorta of E18 and 2-week-old rat. A major proportion of the angiotensin receptors expressed in the aorta at these two ages was AT2 (84 and 81% respectively). Conversely, in the aorta of 8-week-old rats, AT1 was the predominant angiotensin receptor subtype (71%). In 8-week-old rats, the AT2 subtype was also present (28%). In pre- and postnatal rats, [125I]Sar1-angiotensin II binding to AT1 receptors was sensitive to GTPγS whereas binding to AT2 receptors was not. AT2 receptors may serve an important role during stages of rapid growth of the aorta, and also have a significant function in the adult vasculature.

Quantitative autoradiography using the agonist 125I-Sar1-angiotensin II was used to localize and characterize angiotensin II (AT) receptors in the anterior cerebral artery of the male rat. This artery showed a moderately high number of AT receptors, localized throughout the arterial wall. The number of receptors was higher (125 +/- 7 fmol/mg protein) in arteries from young 2-wk-old rats compared with those in adult 8-wk-old rats (43 +/- 2 fmol/mg protein). In the anterior cerebral artery, AT binding was insensitive to displacement with the selective AT1 antagonist DuP 753 but was readily displaced by the selective AT2 antagonist CGP-42112 A (concentration eliciting 50% of maximum inhibition: 6 +/- 1 x 10(-10) M). This indicated that the AT receptors in the cerebral artery were of the AT2 subtype. Our observations suggest that AT may exert its effects on cerebral circulation by stimulation of AT2 receptors and that these receptors may play a role during cerebrovascular development.

Quantitative autoradiography revealed large numbers of angiotensin-II (AT) receptors in the 18-day-old rat embryo. The selective AT-1 antagonist DuP 753 readily competed for AT receptors in liver, lung parenchyma, and choroid plexus, and these receptors are classified as AT-1 receptors. The selective AT-2 displacers CGP 42112 A and/or PD 123177 competed with high affinity with AT bound to most receptors located in skeletal muscle, skin, diaphragm, bronchi, and stomach, and these receptors are classified as AT-2 receptors. The amount of AT-2 receptors in fetal tissue was more than 10-fold higher than that of AT-1 receptors. In skeletal muscle and skin, DuP 753 competed with AT in the presence of 10(-7) M CGP 42112 A, indicating the presence of small numbers of AT-1 receptors. In liver and lung parenchyma, binding to AT-1 receptors was sensitive to guanine nucleotides. AT binding to AT-2 receptors in fetal skin and skeletal muscle was insensitive to guanine nucleotides. AT stimulated phosphoinositide hydrolysis in liver (ED50, 64 nM) and in skin and skeletal muscle (ED50, 62 nM); this was inhibited by DuP 753 (liver IC50, 38 nM; skin and skeletal muscle IC50, 26 nM), but not by PD 123177 in concentrations up to the micromolar range. AT-1 receptors are probably coupled to G-proteins, and their stimulation increases phosphoinositide hydrolysis. AT-2 receptors may not be linked to G-proteins, their stimulation is not associated with phosphoinositide hydrolysis, and the nature of their second messenger system(s) is presently unknown.

Brain angiotensin II receptors were located in 18-day-old rat embryos by quantitative autoradiography. In the nucleus of the solitary tract and choroid plexus, binding was displaced by the type-1 antagonist DuP 753. In the inferior olive, paratrigeminal and hypoglossal nuclei, binding was displaced by the type-2 antagonist CGP 42112 A. Meninges and cephalic soft tissues contained predominantly type-2 angiotensin II receptors. Our results indicate a role for type-1 and type-2 angiotensin II receptors during brain development.

Angiotensin-converting enzyme (ACE, kininase II, EC 3.4.15.1) was quantified in selected areas of the rat brain by 3 different methods. The enzyme activity was quantified by an enzymatic assay in homogenates of discrete brain areas with the use of the artificial substrate hippuryl-His-Leu-[14C]glycine. Binding of the specific ACE inhibitor [125I]351A was quantified after incubation of thin brain sections followed by quantitative autoradiography with comparison to [125I]standards. The antigenic sites of the enzyme were quantified by immunoautoradiography, using a polyclonal antibody to ACE in combination with [125I]protein A followed by quantitative autoradiography. There was a good correlation between the ACE activity, as determined by the quantitative enzymatic method, and both the enzyme inhibitor binding and the [125I]protein A binding, as determined by quantitative autoradiography, in brain areas and in anterior and posterior lobes of the pituitary gland. The present data demonstrate that ACE activity and ACE binding can be quantified in discrete tissue areas with different methods in a variety of conditions and with a high degree of accuracy.

1. Angiotensin II receptors have been studied by quantitative autoradiography in selected brain areas of young (2-week-old) and adult (8-week-old) rats. 2. In young rats, angiotensin II receptors were present in brain areas which did not express receptors in the adult brain, such as thalamic nuclei, cortical areas, and the cerebellum. 3. Young rats had more angiotensin II receptors in the subfornical organ than adult rats. In the inferior olive, the number of angiotensin receptors in young animals was 10 times higher than that in adult rats. Angiotensin II binding in the inferior olive was insentive to incubation in the presence of dithiothreitol. 4. Conversely, the number of angiotensin II receptors in the nucleus of the solitary tract was lower in young rats compared to adults. Incubation in the presence of dithiothreitol resulted in a more than 90% inhibition of angiotensin II binding in the nucleus of the solitary tract. 5. Our results indicate the presence of two types of angiotensin II receptor in brain, one sensitive (type 1) and one insensitive (type 2) to the reducing agent dithiothreitol. 6. The expression of type 2 angiotensin II receptors, insensitive to dithiothreitol, is more marked in young rats, indicating a role for this type of angiotensin receptors in brain development.

We determined beta-adrenoceptor concentrations in sympathetic ganglia from adult Spontaneously Hypertensive Rats (SHR) and Wistar-Kyoto (WKY) control rats by quantitative autoradiography. Adjacent tissue sections were incubated with [125I]iodocyanopindolol, with or without excess of unlabeled (-)-propranolol, the beta 1-antagonist CGP 20712A or the beta 2-antagonist ICI 118,551. Most beta-adrenoceptors were of the beta 2-type. Their concentration was higher in the superior cervical and stellate ganglia of SHR when compared to normotensive WKY. Our results indicate that beta 2-adrenoceptor stimulation may be enhanced in sympathetic ganglia of SHR, and could play a role in the maintenance of their increased sympathetic activity.

The chick retinal melatonin receptor was localized to the inner plexiform layer using 2-125I-melatonin and in vitro autoradiographic techniques. This receptor was of high affinity (Kd19.8 pM) and specificity and showed guanine nucleotide modulation of agonist binding . It thus behaves similarly to other recently characterized high affinity melatonin receptors.

We studied the effects of cations on the binding of the melatonin (MT) agonist, 125I-MT, to MT receptors in the rat area postrema (AP) and suprachiasmatic nuclei (SCN), by using quantitative autoradiography in vitro. Ca2+ promoted agonist binding in the SCN but was without effect in the AP. Na+ induced a dose-dependent loss of agonist binding in both areas. This effect was more drastic in the SCN and also in the absence of divalent cations. The presence of 0.1–4.0 mM Ca2+ or Mg2+ partially and nonselectively reversed this Na+-elicited inhibition. The data agree with known cationic effects on agonist binding to other G protein-coupled receptors and deepen our understanding of the mammalian MT receptor.

Acute immobilization stress increased serotonin and 5-hydroxyindoleacetic acid levels, the 5-hydroxyindoleacetic/serotonin ratio, and the number of [ 3 H]ketanserin binding sites, representing serotonin-2 type receptors, in the rat frontal cortex. Peripheral administration of propranolol or central administration of 6-hydroxydopamine abolished the stress induced elevation of [ 3 H]ketanserin binding sites. Treatment with 6-hydroxydopamine did not affect the increase in serotonin and 5-hydroxyindoleacetic acid levels, and enhanced the increase in the 5-hydroxyindoleacetic acid/serotonin ratio produced by stress. Conversely, chemical serotoninergic denervation with 5,7-dihydroxytryptamine had no influence on the stress-induced elevation of [ 3 H]ketanserin binding sites, but abolished the serotonin and 5-hydroxyindoleacetic acid increase produced by stress. These results suggest that an intact serotoninergic system is not essential for serotonin-2 type receptor regulation during stress. Instead, the noradrenergic system, most probably through stimulation of β-adrenoreceptors, may control the regulation of [ 3 H]ketanserin binding sites in the rat frontal cortex during acute stress.

We studied the phosphoinositide (PI) turnover in the pineal gland of spontaneously hypertensive rats (SHR) and compared it to that in Wistar-Kyoto and Wistar rats. Both basal and norepinephrine-stimulated PI turnover were significantly higher in the SHR than in the other strains. Prazosin (1 mumol/L) completely blocked the norepinephrine-induced PI hydrolysis in all groups. However, prazosin failed to lower the enhanced basal PI turnover in SHR. Pineal response to the cholinergic agonist carbachol was similar in all groups. These results suggest enhanced membrane phospholipid turnover in the pineal gland of SHR. Moreover, signaling via the alpha 1-adrenoceptor might be sensitized in the SHR.

We have characterized melatonin (MT) receptors in the rat suprachiasmatic nuclei by using quantitative autoradiography in vitro at equilibrium conditions for a picomolar affinity site. Binding of the MT agonist 2-[125I]iodomelatonin (30 pM to 6 nM) was saturable, of high affinity (Kd, 52.8 pM), of high specificity, and to a single class of sites (binding capacity, 16.5 fmol/mg protein). However, by shortening the washing time for bound and free ligand, we were able to demonstrate an additional low affinity form of this receptor (Kd, 761 pM; binding capacity, 72.2 fmol/mg protein). Micromolar concentrations of guanine nucleotides and millimolar concentrations of monovalent cations (Na+ and Li+) dose-dependently and specifically inhibited agonist binding at 22 C. Saturation studies revealed that this was due to a decrease in receptor affinity in both cases. Ca+2 promoted high affinity agonist binding, as omission of this cation decreased the affinity of the suprachiasmatic MT receptor. These results suggest coupling of the rat suprachiasmatic MT receptors to guanine nucleotide-binding regulatory protein(s). Moreover, our data demonstrate specific modulation of the affinity of these receptors with cations.

Angiotensin-converting enzyme (ACE, kininase II, EC 3.4.15.1) was visualized in unfixed rat brain sections by autoradiography after incubation with a polyclonal goat anti-rabbit lung converting-enzyme antibody and [125I]protein A. Paraformaldehyde fixation interfered with the recognition of ACE by its antibody in brain nuclei but not in the choroid plexus. Conversely, incubation of brain sections with the specific ACE inhibitor [125I]351A allowed ACE visualization in either unfixed and paraformaldehyde-fixed brain sections, since [125I]351A binding was not affected by our fixation conditions. Our results indicate that ACE could be visualized in unfixed brain sections directly by incubation with the specific inhibitor [125I]351A or indirectly by radioimmunohistochemistry and autoradiography. Different autoradiographic methods could be used to visualize and quantify ACE in unfixed or fixed tissues.

ACTIVATION OF BRAIN ANP RECEPTORS DURING CHRONIC ECF VOLUME CONTRACTION IN YOUNG RATS. † 2192

We report a simple two-step immunocytochemical method that uses staphylococcal protein A conjugated to horseradish peroxidase and the H2O2-3,3'-diaminobenzidine chromogenic reaction to stain neurons and fibers containing immunoreactive cell antigens. Our protein A-peroxidase method was found to produce stains of equal or superior quality to those of the classical 3-step peroxidase-anti-peroxidase (PAP) technique. Since the chromogenic reaction with protein A-peroxidase has low non-specific tissue binding, the reaction can be carried out at acidic pH with higher horseradish peroxidase activity and no significant increase in background. Using this method, we were able to produce clear stains of neurons and fibers containing tyrosine hydroxylase or fibers containing methionine-enkephalin. The use of protein A-peroxidase conjugate substantially simplified the immunocytochemistry procedure, and reduced both the time and cost of experiments.

The β-adrenoceptor antagonist pindolol, given p.o. (10 mg/kg, once a day for 7 days) to 4 and 24 week old male spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY), decreased plasma angiotensin-converting enzyme (ACE) activity only in young SHR. Kinetic studies indicated that the treatment reduced the ACE maximal velocity or the number of available ACE molecules. In vitro studies showed that pindolol had no direct effects on the activity of plasma ACE. A decrease in soluble (loosely bound) ACE activity was observed in the lung of young SHR treated with pindolol. The results suggest that pindolol affects the site of origin of plasma ACE. The susceptibility of young SHR to pindolol may indicate a probable role of β-adrenoceptors in the regulation of ACE activity in spontaneous (genetic) hypertension.

By immunocytochemical techniques, the neuronal cell bodies containing ACTH, β-LPH and α-MSH have only been found in the area of the arcuate nucleus of the hypothalamus, whereas positive nerve fibers have been observed in many hypothalamic and extra-hypothalamic areas. The possible contribution of neurons which could be located in other brain areas has been studied in the rat by experiments involving hypothalamic deafferentation or destruction of the acurate nucleus. Fourteen days after deafferentation, no immunoreactive fibers could be detected in extrahypothalamic areas whereas the concentration of positive cell bodies and fibers remained unchanged within the hypothalamic island. In rats which had been injected in the neonatal period with monosodium glutamate (MSG), which selectively destroys the arcuate nucleus, only a few immunostained cell bodies were observed in hypothalamic region lateral to the arcuate nucleus. As compared to control animals, the concentration of immunostained fibers in both hypothalamic and extrahypothalamic regions was markedly decreased. These results strongly suggest that neuronal cell bodies producing ACTH, β-LPH and α-MSH are located in the region of the arcuate nucleus and send axonal projections into many brain areas.

Angiotensin II (Ang) binding kinetics were determined in discrete brainstem nuclei of 14-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) by a quantitative autoradiographic technique. Tissue sections were incubated with 125I-labeled [sarcosine-1]Ang, and results were analyzed by computerized densitometry and comparison to 125I-labeled standards. A single class of high-affinity binding sites was identified in the nucleus tractus solitarius, the area postrema, and the inferior olivary nuclei of both SHR and WKY rats. Ang binding affinity was significantly greater in the nucleus tractus solitarius of SHR compared to normotensive WKY rats (0.27 +/- 0.06 X 10(9) M-1 in WKY rats vs. 0.59 +/- 0.15 X 10(9) M-1 in SHR), with no apparent changes in the maximum binding capacity of this area. There were no changes in the Ang binding kinetics of the area postrema or the inferior olivary nuclei. Our results suggest that central Ang activity is altered in established hypertension in a brainstem area of SHR associated with peripheral cardiovascular control.

1. Acute immobilization stress results in an increase in pineal adrenaline levels, which is prevented by prior removal of the adrenal medulla, indicating that the adrenal medulla is the primary source of elevated adrenaline concentrations in the pineal gland during stress. 2. Adrenomedullectomy does not result in a decrease in the low pineal adrenaline levels observed in nonstressed rats. This finding, together with the presence in the pineal gland of the adrenaline-forming enzyme, phenylethanolamine-N-methyltransferase (PNMT; EC 2.1.1.28), indicates that local synthesis of small amounts of adrenaline can normally occur in the pineal gland. 3. The lack of change in methylating activity after denervation of the pineal gland suggests that adrenaline synthesis may normally occur postsynaptically. 4. No significant changes occur in the pineal concentrations of noradrenaline and dopamine after stress and adrenomedullectomy, with the exception of increased dopamine levels after stress in adrenomedullectomized rats. 5. Adrenaline concentrations show a 24-hr rhythm in the rat pineal gland, with maximum levels during the second half of the dark period (0100 to 0500 hr) and low concentrations at the end of the light period (1700 hr). The changes in pineal adrenaline levels parallel those of the other catecholamines, dopamine and noradrenaline, indicating that increased synthesis and/or uptake from the circulation of all three catecholamines can occur synchronously during the latter part of the dark period. 6. Our results suggest a physiological role for pineal adrenaline, together with dopamine and noradrenaline, in the regulation of the daily pineal rhythms and during stress.

1. We have studied the localization, kinetics, and regulation of receptors for the circulating form of the atrial natriuretic peptide (ANP; 99–126) in the rat brain. 2. Quantitative autoradiographic techniques and a125I-labeled ligand,125I-ANP (99–126), were employed. Afterin vitro autoradiography, quantification was achieved by computerized microdensitometry followed by comparison with125I-standards. 3. ANP receptors were discretely localized in the rat brain, with the highest concentrations in circumventricular organs, the choroid plexus, and selected hypothalamic nuclei involved in the production of the antidiuretic hormone vasopressin and in blood-pressure control. 4. Spontaneously (genetic) hypertensive rats showed much lower numbers of ANP receptors than normotensive controls in the subfornical organ, the area postrema, the nucleus of the solitary tract, and the choroid plexus. These changes are in contrast to those observed for receptors of angiotensin II, another circulating peptide with actions opposite to those of ANP. 5. Under conditions of acute dehydration after water deprivation, as well as under conditions of chronic dehydration such as those present in homozygous Brattleboro rats, there was an up-regulation of ANP receptors in the subfornical organ. 6. Our results indicate that in the brain, circumventricular organs contain ANP receptors which could respond to variations in the concentration of circulating ANP. In addition, brain areas inside the blood-brain barrier contain ANP receptors probably related to the endogenous, central ANP system. 7. The localization of ANP receptors and the alterations in their regulation present in genetically hypertensive rats and after dehydration indicate that brain ANP receptors are probably related to fluid regulation, including the secretion of vasopressin, and to cardiovascular function. ANP and angiotensin II could act as mutual antagonists in the brain as they do in the periphery. 8. ANP receptors in the choroid plexus may be related to the formation of cerebrospinal fluid.

A single class of high-affinity binding sites for [125I]angiotensin III and [125I]angiotensin II were found in rat adrenal medulla and zona glomerulosa by quantitative autoradiography. In the medulla, Kd were 1.46 and 1.16 nM, and Bmax 1700 and 1700 fmol/mg protein, for [125I]angiotensin II and [125I]angiotensin III, respectively. In the zona glomerulosa, Kd were 0.86 and 0.90 nM, and Bmax 790 and 560 fmol/mg protein, for [125I]angiotensin II and [125I]angiotensin III, respectively. Unlabeled angiotensin III and angiotensin II displaced [125I]angiotensin III with similar potency in both adrenal zona glomerulosa and medulla. Our findings suggest that angiotensin III and angiotensin II might share the same binding sites in adrenal gland and support the hypothesis of a role for angiotensin III in the adrenal medulla and zona glomerulosa.

1. In pineal glands of adult spontaneously hypertensive rats (Okamoto strain), dopamine levels increased 34%, adrenaline concentrations increased 152% and noradrenaline levels decreased by 26%, when compared with Wistar-Kyoto normotensive controls. 2. These results are consistent with the hypothesis of an increased uptake of peripheral adrenaline resulting in increased release of noradrenaline from pineal sympathetic nerves in hypertensive rats. 3. The activity of pineal N-acetyltransferase was increased fourfold in hypertensive rats, indicating increased stimulation of pineal β-adrenoreceptors. 4. Methylation of pineal membrane phospholipids was also increased (100-320%) in hypertensive rats. 5. These results indicate a correlation in vivo between increased β-adrenoreceptor stimulation and increased methylation of membrane phospholipids in the rat pineal gland, which could result in changes in membrane fluidity and affect the coupling of the β-adrenoreceptors to the adenylate cyclase.

Atrial natriuretic peptide (ANP) binding sites were measured by quantitative autoradiography in the supraoptic and paraventricular nuclei and in the subfornical organ of hypophysectomized, adrenalectomized, and genetically vasopressin-deficient (Brattleboro) rats. Hypophysectomized and Brattleboro rats had significantly higher numbers of ANP binding sites in the supraoptic nucleus and in the magnocellular subdivision of the paraventricular nucleus than their respective controls. ANP binding density was also increased in the parvocellular subdivision of the paraventricular nucleus in hypophysectomized rats and in the subfornical organ of homozygous Brattleboro rats. When homozygous Brattleboro rats were treated with vasopressin, the density of ANP binding sites was restored to control level in the subfornical organ but not in the supraoptic or paraventricular nuclei. Adrenalectomy did not influence ANP binding in the brain areas studied. Increased ANP binding density in Brattleboro rats and after hypophysectomy in the nuclei in which vasopressin neurons are located suggest that ANP binding sites may represent physiologically active receptors and may mediate the inhibitory action of ANP on vasopressin secretion.

beta-Adrenoceptors were localized and characterized in valve leaflets of the rat heart. Sixteen micrometer-thick tissue sections containing the mitral and aortic valves were incubated with (-)3-[125I]iodocyanopindolol followed by autoradiography with computerized microdensitometry and comparison with 125I-labeled standards. beta-Adrenoceptors were present in all the valves studied. The selective beta 1-adrenoceptor antagonist CGP 20712 A (100 nM) displaced not more than 20% of the total binding sites, suggesting that most of the beta-adrenoceptors in the valve leaflets are of the beta 2-subtype. Forskolin-binding sites were detected in the mitral valve leaflet by incubation of adjacent tissue sections with [12-3H]forskolin. Our results indicate that catecholamines could regulate the function of the heart valves through stimulation of beta 2-adrenoceptors.

Renal alpha 1 and alpha 2-adrenergic receptors were quantified in Dahl salt-sensitive and salt-resistant rats, in Okamoto-Aoki spontaneously hypertensive rats (SHR), in Wistar Kyoto "normotensive" (WKY), and in Charles River rats made hypertensive by the Grollman ligature technique and by DOC-NaCl administration after unilateral nephrectomy. The effect of high dietary NaCl on renal alpha receptors was studied in Dahl, SHR, and WKY rats. Renal alpha 1 and alpha 2 receptor densities were higher (p less than 0.05) in SHR and in Dahl salt-sensitive rats than in their normotensive controls. High dietary sodium increased renal alpha 2 receptors and blood pressure in SHR, WKY and Dahl salt-sensitive, but not in resistant Dahl rats. A study of time relationships revealed that the increase in renal alpha 2 receptors preceded most of the blood pressure elevation due to high dietary sodium. Renal alpha-adrenergic receptor densities of surgical (Grollman) and endocrine (DOC-NaCl) forms of rat hypertension were not different from normotensive controls. Thus, renal alpha 2 receptor density and increase thereof by dietary sodium may be: 1) a biochemical marker for genetic forms of hypertension in the rat, and 2) closely linked to the basic mechanism of high blood pressure.

Carbachol induced a dose-dependent accumulation of inositol monophosphates in the Wistar rat pineal gland. The relative potency of muscarinic antagonists (atropine greater than pirenzepine much greater than gallamine) in blocking this response suggests the involvement of muscarinic M1 receptors. Following bilateral superior cervical ganglionectomy, the carbachol-induced phosphoinositide response was similar to that seen in sham-operated controls, while adrenergic response was enhanced by 50%. The results indicate that the pineal has functional muscarinic receptors which are not affected by removal of the sympathetic innervation to the gland.

1. We have used horseradish peroxidase-conjugated protein A- and125I-protein A to develop immunohistochemical and radioimmunohistochemical methods for the localization of antigens in brain and other tissues of the rat. 2. We visualized methionine-enkephalin fibers in the rat brain by incubating tissue sections with a specific polyclonal antibody and peroxidase-conjugated protein A. The method is simple, fast, and less expensive and more sensitive than classical immunohistochemical techniques and the principle could be used to visualize many other tissue antigens. 3. Incubation of tissue samples with specific polyclonal antibodies and125I-protein A, followed by autoradiography, allows the permanent recording of the radiommunohistochemical localization of brain methionine-enkephalin, tyrosine hydroxylase, and angiotensin-converting enzyme and of pituitary vasopressin and could be applied to the localization of many other tissue antigens. 4. A new quantitative radioimmunohistochemical technique for methionine-enkephalin allows the determination of the endogenous peptide content in discrete brain nuclei from 16-µm-thick sections. The method is based on the quantitative determination of the amount of125I-protein A bound to specific tissue areas after incubation with a specific polyclonal antibody, followed by autoradiography and computerized microdensitometry. To quantify the endogenous peptide content, the values obtained are interpolated into a methionine-enkephalin internal standard curve. This standard curve was constructed by measuring endogenous concentrations of methionine-enkephalin by radioimmunoassay in specific brain regions and correlating these values with quantitative autoradiographic determinations in homologous areas of adjacent sections. Similar methods can be developed for other tissue antigens. 5. These new methods allow for the localization and quantification of tissue antigens in very discrete areas of the brain and other tissues and have a wide application in neurobiology and pathology.

Exogenous arachidonic acid (AA) incubated in presence of male rat hypothalamus, shows a low rate of conversion (less than 1%) of the substrate with a major product, identified as 12-hydroxyeicosatetraenoic acid (12-HETE) by reverse phase-high performance liquid chromatography (rpHPLC) and gas chromatography-mass spectrometry (GC-MS). Furthermore, immunoreactive 12-HETE estimated after purification on rpHPLC is produced by hypothalamus slices or median eminences (MEs) incubated in absence of any exogenous precursor. The effect of 12-HETE was tested on the release of LHRH from rat MEs after a 30-min incubation and was compared to the effect of another lipoxygenase product, 5-HETE, and to the well-known stimulatory effect of prostaglandin E2 (PGE2). The three AA metabolites stimulate LHRH release. A significant stimulatory effect on LHRH release is obtained with 10(-9) M of 12-HETE and only with 10(-8) M of 5-HETE or PGE2. Furthermore, the effect of higher concentrations is different according to the eicosanoid tested. The maximal response (176% of the control) is reached with 12-HETE at 10(-8) M. No significant change is observed at 10(-7) and 10(-6) M. The response with 5-HETE is also maximal (162% of the control) at 10(-8) M but decreases significantly (only 117% of the control) at 10(-6) M. The amplitude of the response to PGE2 is larger and higher, reaching a plateau (300% of the control) at 10(-6) M. 12-HETE has no effect on somatostatin (SRIF), release, as already known for PGE2.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin-converting enzyme (ACE, kininase II, EC 3.4.15.1) activity was measured in forebrain nuclei of 4- and 16-week-old spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) normotensive controls. Young SHR showed lower enzyme activity in the nucleus paraventricularis, and higher activity in the nuclei septalis medialis and habenularis medialis. Adult SHR exhibited lower ACE activity in the nucleus septalis medialis, the organon subfornicalis, the organon vasculosum laminae terminalis, and the nuclei periventricularis and suprachiasmatis.