Autoradiographic localization of angiotensin II receptors in rat brain.

The 125I-labeled agonist analog [1-sarcosine]-angiotensin II ( [Sar1]AII) bound with high specificity and affinity (Ka = 2 X 10(9) M-1) to a single class of receptor sites in rat brain. This ligand was used to analyze the distribution of AII receptors in rat brain by in vitro autoradiography followed by computerized densitometry and color coding. A very high density of AII receptors was found in the subfornical organ, paraventricular and periventricular nuclei of the hypothalamus, nucleus of the tractus solitarius, and area postrema. A high concentration of receptors was found in the suprachiasmatic nucleus of the hypothalamus, lateral olfactory tracts, nuclei of the accessory and lateral olfactory tracts, triangular septal nucleus, subthalamic nucleus, locus coeruleus, and inferior olivary nuclei. Moderate receptor concentrations were found in the organum vasculosum of the lamina terminalis, median preoptic nucleus, medial habenular nucleus, lateral septum, ventroposterior thalamic nucleus, median eminence, medial geniculate nucleus, superior colliculus, subiculum, pre- and parasubiculum, and spinal trigeminal tract. Low concentrations of sites were seen in caudate-putamen, nucleus accumbens, amygdala, and gray matter of the spinal cord. These studies have demonstrated that AII receptors are distributed in a highly characteristic anatomical pattern in the brain. The high concentrations of AII receptors at numerous physiologically relevant sites are consistent with the emerging evidence for multiple roles of AII as a neuropeptide in the central nervous system.     

Atrial natriuretic factor: localization in the adrenal gland of the lizard Podarcis sicula and effects on pituitary-adrenal axis activity

The occurrence of atrial natriuretic factor (ANF) immunoreactivity was investigated in the adrenal gland of the lizard Podarcis sicula by avidin-biotinylated peroxidase complex (ABC) immunocytochemical technique: ANF immunoreactivity was present in the chromaffin tissue, and was absent in the steroidogenic tissue. The role of ANF in the modulation of the pituitary-adrenal axis activity was investigated in vivo by intraperitoneal administration of ANF. The effects were evaluated by examination of the morphological and morphometrical features of the tissues, as well as the plasma levels of adrenocorticotropic hormone (ACTH), corticosterone, aldosterone, norepinephrine, and epinephrine. ANF (28 microg/100 g body wt) did not affect ACTH plasma levels, that remained almost unchanged; in contrast, corticosterone plasma levels increased from 6.45 +/- 0.070 ng/ml in carrier-injected lizards to 9.69 +/- 0.080 ng/ml 24 h after the injection; aldosterone levels decreased from 2.19 +/- 0.010 ng/ml in carrier-injected specimens to 0.58 +/- 0.003 ng/ml 24 h after the experimental treatment. In the chromaffin tissue, an increase in the number of epinephrine cells and a decrease in the number of norepinephrine cells were observed, decreasing the numeric norepinephrine/epinephrine cell ratio, from 1.4/1 of control specimens to 0.3/1 24 h after ANF administration. Moreover, norepinephrine plasma levels decreased from 998 +/- 4.600 pg/ml in carrier-injected specimens to 321 +/- 2.230 pg/ml 24 h after ANF administration; epinephrine plasma levels were elevated from 614 +/- 3.410 pg/ml in carrier-injected specimens to 1672 +/- 10.800 pg/ml 24 h after the experimental treatment. The presence of ANF in the adrenal gland suggests that, also in reptiles as in other vertebrates, this peptide, locally released from the chromaffin cells, may modulate the activity of the adrenal gland, probably in a paracrine manner. The effects of ANF on the adrenal gland suggest that this peptide may affect reptilian salt and fluid homeostasis.     

Vascular and adrenal receptors for atrial natriuretic factor in the rat

Previous studies have shown that atrial natriuretic factor, a powerful vasorelaxant of precontracted vessels, inhibits the secretion of aldosterone stimulated by angiotensin II, adrenocorticotropic hormone, and potassium. We now report the presence of specific binding sites for atrial natriuretic factor in rat blood vessels (mesenteric and renal arteries) and adrenal capsules. Radioiodinated synthetic atrial natriuretic factor bound to a single class of high-affinity (KD = 0.1 nM) low-capacity receptors in a particulate fraction from blood vessels and adrenals. Unrelated peptides did not displace atrial natriuretic factor. Fragments of atrial natriuretic factor displaced the labeled ligand with decreasing potency after cleavage at the N-terminal. The cleavage of the C-terminal tyrosine did not decrease the potency of atrial natriuretic factor, but further cleavage at the C-terminal dramatically reduced the affinity of the resulting peptides. The potency of the atrial natriuretic factor fragments in the radioligand assay was in proportion to their potency to inhibit aldosterone secretion by isolated rat glomerulosa cells. Our results suggest that these binding sites mediate the biological actions of atrial natriuretic factor in blood vessels and the adrenal, and that both receptors have similar specificities.     

Autoradiographic localization of beta-adrenergic receptors in rat oviduct

Catecholamines are known to have an inhibitory effect on oviductal smooth musculature by changing the adrenergic receptors activity. In order to further investigate the role of epinephrine and/or norepinephrine in oviduct, the distribution of beta-adrenergic receptors has been studied in the rat oviduct, using in vitro autoradiography and [125I]cyanopindolol (CYP), as radioligand. The specificity of the labelling and the characterization of receptor subtypes in different cell types was achieved by displacement of radioligand with increasing concentrations of zinterol, a beta-adrenergic agonist with preferential affinity for the beta 2-adrenoreceptor subtype and practolol, a beta-adrenergic antagonist that binds preferentially to beta 1-subtype. Quantitative estimation of ligand binding was achieved by densitometry. It was shown that the vast majority of beta-receptors were of the beta 2-subtype and were found in smooth muscle layers as well as in the epithelium. The latter localization suggests a role for epinephrine and/or norepinephrine on the oviductal epithelium.     

Autoradiographic localization of [125I]-angiotensin II binding sites in the rat adrenal gland

To gain greater insight into sites of action of circulating angiotensin II (Ang II) within the adrenal, we have localized the [125I]-Ang II binding site using in vitro autoradiography. Autoradiograms were generated either by apposition of isotope-sensitive film or with emulsion-coated coverslips to slide-mounted adrenal sections labeled in vitro with 1.0 nM [125I]-Ang II. Analysis of the autoradiograms showed that Ang II binding sites were concentrated in a thin band in the outer cortex (over the cells of the zona glomerulosa) and in the adrenal medulla, which at higher power was seen as dense patches. Few sites were evident in the inner cortex. The existence of Ang II binding sites in the adrenal medulla was confirmed by conventional homogenate binding techniques which revealed a single class of high affinity Ang II binding site (Kd = 0.7nM, Bmax = 168.7 fmol/mg). These results suggest that the adrenal medulla may be a target for direct receptor-mediated actions of Ang II.     

Atrial natriuretic factor modulates proximal glomerulotubular balance in anesthetized rats

The extent to which the natriuretic effect of a prolonged low dose infusion of atrial natriuretic factor (30 ng/kg/min) is dependent on interference with the prevailing intrarenal actions of angiotensin II was examined before and after blockade of angiotensin production with the converting enzyme inhibitor enalaprilat (5 mg/kg). Lithium clearance was used to assess proximal tubular sodium and water reabsorption. Atrial natriuretic factor and enalaprilat caused similar increases in sodium excretion (10-fold and sevenfold, respectively) and glomerular filtration rate (each 34%) and similar decreases in fractional proximal reabsorption of sodium (17% and 13%, respectively) and blood pressure. Each also caused a major disruption in the effectiveness of proximal glomerulotubular balance (30% and 50% of perfect balance). Infusion of atrial natriuretic factor during converting enzyme inhibition increased glomerular filtration rate further by 23%, reaching 63% above control without change in renal blood flow but with a rise in filtration fraction to 0.48. Sodium excretion increased further but fractional proximal sodium reabsorption remained constant and proximal glomerulotubular balance appeared to improve. Atrial natriuretic factor therefore possesses a glomerular action that persists during converting enzyme inhibition and is indeed additive to the removal of angiotensin II when the proximal effect of atrial natriuretic factor is no longer apparent. It is concluded that failure of atrial natriuretic factor to further suppress fractional proximal sodium reabsorption during converting enzyme inhibition is caused by either prior removal of the stimulatory action of angiotensin II on proximal tubular transport or extreme changes in peritubular physical factors consequent on the high filtration fraction.     

Synthesis, internalization, and localization of atrial natriuretic peptide in rat adrenal medulla

Some, though not all studies, have indicated that atrial natriuretic peptide (ANP) can bind to adrenal medullary cells. ANP-like immunoreactivity (ANP-LI) has also been identified in catecholamine-secreting cells. Together, these findings suggest that ANP may be taken up and/or synthesized in the adrenal medulla. The present study was designed to ascertain, by in situ hybridization, whether adrenal chromaffin cells could synthesize ANP, to define by an in vivo ultrastructural autoradiographic approach, whether ANP could, in fact, bind to rat adrenal medulla cells, to determine whether there was a cellular [noradrenaline (NA) vs. adrenaline (A)] selectivity in the binding process, and to establish whether extracellular [125I]ANP could be internalized by these cells. The cellular and subcellular distribution of endogenous ANP-LI was also investigated in both cell types by cryoultramicrotomy and immunocytochemical approaches. The in situ hybridization studies indicate the presence of mRNA to ANP in about 15% of adrenal medullary cells. Intravenous injection of [125I]ANP resulted in a 3-fold, preferential and specific radiolabeling of A-as compared to NA-containing cells. In A-containing cells, plasma membranes were significantly labeled 2 and 5 min post injection; cytoplasmic matrix, mitochondria, and secretory granules throughout the time course studied (1-30 min post injection). Lysosomes, rough endoplasmic reticulum, Golgi apparatus, and nuclei were not labeled. ANP-LI was identified in both NA- and A-containing cells; in the former, it was almost exclusively localized in secretory vesicles, in the latter it was detected in plasma membranes, cytoplasmic matrix, nuclear euchromatin, some mitochondria and relatively fewer granules than in NA-containing cells. The findings suggest that ANP may be synthesized primarily in NA-containing cells and that A-containing cells primarily bind and internalize the extracellular (endogenous or exogenous) atrial peptide. The data suggest that ANP secreted by adrenal medullary chromaffin cells may have distal paracrine actions or interactions with coreleased catecholamines and neuropeptides. Binding and internalization may reflect an action of ANP on the secretory function of A-containing cells.     

Corticotropin-releasing factor receptors in human pituitary gland: autoradiographic localization

Corticotropin-releasing factor (CRF) receptors were localized in the human pituitary gland by an in vitro labeling light microscopic autoradiographic technique using an 125I-labeled analog of ovine CRF substituted with norleucine and tyrosine at amino acid residues 21 and 32, respectively. Specific binding sites for CRF were observed in the anterior lobe while no specific binding sites for CRF were present in the posterior pituitary lobe. The CRF binding sites in the anterior lobe occurred in clusters which predominated in the anteromedial portion of the lobe. This pattern of localization of CRF binding sites resembles previously reported distributions of corticotrophs in anterior pituitary. These data support the physiological role of endogenous CRF in regulating hormone secretion from the anterior lobe of the human pituitary.     

Autoradiographic localization of relaxin binding sites in rat brain.

Relaxin is a member of the insulin family of polypeptide hormones and exerts its best understood actions in the mammalian reproductive system. Using a biologically active 32P-labeled human relaxin, we have previously shown by in vitro autoradiography specific relaxin binding sites in rat uterus, cervix, and brain tissues. Using the same approach, we describe here a detailed localization of human relaxin binding sites in the rat brain. Displaceable relaxin binding sites are distributed in discrete regions of the olfactory system, neocortex, hypothalamus, hippocampus, thalamus, amygdala, midbrain, and medulla of the male and female rat brain. Characterization of the relaxin binding sites in the subfornical organ and neocortex reveals a single class of high-affinity sites (Kd = 1.4 nM) in both regions. The binding of relaxin to two of the circumventricular organs (subfornical organ and organum vasculosum of the lamina terminalis) and the neurosecretory magnocellular hypothalamic nuclei (i.e., paraventricular and supraoptic nuclei) provides the anatomical and biochemical basis for emerging physiological evidence suggesting a central role for relaxin in the control of blood pressure and hormone release. We conclude that specific, high-affinity relaxin binding sites are present in discrete regions of the rat brain and that the distribution of some of these sites may be consistent with a role for relaxin in control of vascular volume and blood pressure.     

Atrial natriuretic factor plays a significant role in body fluid homeostasis

Atrial natriuretic factor (ANF) is a hormone with the physiological characteristics of a regulator of body fluid volume. It is potent, has a short duration of action, and responds to a physiologically relevant stimulus in a negative feedback-controlled system. It can act directly or indirectly (via inhibition of aldosterone biosynthesis) on the kidney to alter sodium transport and may regulate fluid distribution within the extracellular space. The peptide circulates at low (nanomolar) levels, and recent studies with renal inner medullary cells document relevant receptor binding and second messenger activation in this concentration range. In vivo data support a direct action on the kidney to enhance natriuresis, and blockade of a primary catabolic pathway for ANF within the kidney results in augmented natriuresis at concurrent endogenous peptide concentrations. Long-term, low dose infusion directly into the renal artery of conscious dogs supports a physiological action of ANF to promote urinary sodium excretion. Nevertheless, under certain circumstances, natriuresis does not occur even at high circulating levels of ANF. Apparently other factors such as renal perfusion pressure, volume status, and renal nerve activity are important in determining the natriuretic response to a given level of peptide. We hypothesize that the role played by ANF in volume regulation is highly complex, and the kidney responds with increased sodium excretion only when a constellation of variables is appropriately arrayed. That is, ANF is a necessary, but not sufficient, condition to induce natriuresis.     

Autoradiographic localization of binding sites for oxytocin and vasopressin in the rat kidney

The distribution of [3H]vasopressin- and [3H]oxytocin-binding sites was examined, using an autoradiographical technique, in the kidney of Long-Evans and Brattleboro rats. Two types of binding sites with affinities in the nanomolar range were detected: one, located on glomeruli, bound both vasopressin and oxytocin; the other, on collecting ducts, bound vasopressin selectively. In the presence of 10 mumol oxytocin/l, [3H]vasopressin labelling was abolished in glomeruli, but only reduced in collecting ducts; [3H]oxytocin labelling was completely abolished by 10 mumol vasopressin/l. These observations are discussed in relation to known effects of neurohypophysial hormones on renal physiology.     

Peripheral-type benzodiazepine receptors in endocrine organs: autoradiographic localization in rat pituitary, adrenal, and testis

We have used [3H]Ro5-4864, a ligand selective for peripheral-type benzodiazepine receptors, to identify and localize peripheral-type benzodiazepine receptors in endocrine organs. Autoradiographic studies reveal an uniform distribution of [3H]Ro5-4864 binding sites within the anterior, intermediate, and posterior lobes of the pituitary gland, with highest concentrations present in the posterior pituitary. In rat adrenal gland, specific binding sites for [3H]Ro5-4864 are found only in the adrenal cortex, with highest density in the zona glomerulosa and significantly lower concentrations in the zona fasciculata and zona reticularis. [3H]Ro5-4864-associated silver grains in the testis are intensely localized over the interstitial tissue; low concentrations of silver grains are present over the epithelium of the seminiferous tubules but are absent from the tubular lumen. These studies demonstrate a differential and discrete localization of peripheral-type benzodiazepine receptors in rat pituitary, adrenal, and testis.     

Atrial natriuretic factor in specific brain areas of spontaneously hypertensive rats

Atrial natriuretic peptides (atrial natriuretic factor, ANF) are present in a great number of brain areas inside and outside of the blood-brain barrier. The pattern of distribution implies the involvement of ANF in different physiological functions, such as blood pressure regulation, electrolyte and fluid homeostasis, and modulation of the neuroendocrine system. To further investigate a possible involvement of central ANF in spontaneous hypertension, we measured levels of ANF in 18 selected, microdissected brain areas of prehypertensive (4-week-old) and hypertensive (12-week-old) spontaneously hypertensive rats (SHR) and their normotensive control, Wistar-Kyoto rats (WKY), by radio-immunoassay. ANF was significantly decreased in seven brain areas in SHR at both ages investigated; the most pronounced decreases were found in the subfornical organ, in the perifornical and periventricular hypothalamic nuclei, and in the medial preoptic nucleus. In addition, in young SHR ANF was significantly decreased in the organum vasculosum laminae terminalis and increased in the median eminence. After the development of hypertension, a significant decrease of ANF could be detected in four more brain areas (bed nucleus of the stria terminalis, paraventricular and arcuate nuclei, dorsal raphe nucleus) of SHR, as compared with normotensive controls, and the increase in the median eminence was no longer detectable. These results suggest a role for ANF in genetic hypertension and the specific importance of certain brain regions.     

Autoradiographic localization of putative arginine vasotocin receptors in the kidney of a urodele amphibian

The distribution and characteristics of putative arginine vasotocin (AVT) receptors in the urodele amphibian kidney were investigated using in vitro quantitative autoradiography. Specific binding sites for [3H]arginine vasopressin (AVP) in the kidney of the rough-skinned newt (Taricha granulosa) were located over the glomeruli. Scatchard analysis showed that, in the range of concentrations tested (0.2 to 22 nM), [3H]AVP bound to a single class of receptors with a dissociation constant of 1.4 nM and a binding site concentration of 36.5 fmol/mg protein. Binding displacement studies showed that both AVT and AVP were potent ligands for newt kidney receptors. Two specific antagonist peptides with anti-vasopressor (V1) activity, but not anti-antidiuretic (V2) activity, in rat tissues were tested as well. Both antagonists effectively displaced [3H]AVP from receptor sites in newt kidney slices, indicating that the binding sites in this amphibian resemble the V1 subtype of mammals in ligand specificity. Localization of AVT receptors over kidney glomeruli and ligand specificity of these sites is consistent with the hypothesis that AVT may cause antidiuresis in urodele amphibians at least in part via a glomerular vasoconstricting action.     

Autoradiographic localization of corticosterone receptors (type III) to the collecting tubule of the rat kidney.

Recently, a class of receptors exhibiting high affinity for corticosterone was described in rat kidney (Feldman, D. et al., Endocrinology 92: 1429, 1973). These receptor sites exhibited negligible affinity for dexamethasone and aldosterone and were designated Type III to distinguish them from sites having high affinity for aldosterone (Type I), and sites with high affinity for dexamethasone and corticosterone (Type II). To visually localize Type III sites in the kidney and demonstrate whether or not they represent intracellular steroid receptors, we used an autoradiographic procedure for diffusible substances. Male adrenalectomized rats were injected intravenously with the following combination of steroids per 100 g body weight: 4 x 10(-9) mol [3H]corticosterone, 4 x 10(-9) mol unlabeled aldosterone, and 4 x 10(-9) mol unlabeled dexamethasone. To differentiate "nonspecific" binding, each experimental animal was paired with a control animal that received the same steroids plus 250-fold unlabeled corticosterone. At 3 min, 10 min, and 30 min, kidneys were removed, cut into quadrants, and frozen in isopentane cooled by liquid nitrogen. For autoradiography, 4 mum frozen sections were cut, pressed into contact with emulsion precoated slides at -30 C, melted and simultaneously dried under a jet of dry nitrogen gas, and exposed at 4 C for 2 to 6 weeks. At all three time intervals, silver grains representing [3H]corticosterone binding sites, were concentrated over collecting tubules, only in the outer medulla and cortex (those in the inner medulla and papilla were not labeled). In the labeled segments of the nephron, some of the cells showed an apparent high ratio of cytoplasmic to nuclear grains and in others nuclear labeling was more prominent. A small population of cells within labeled collecting tubules (possibly dark cells) were not labeled. Although no function can yet be ascribed to Type III receptors in the kidney, they may represent an important steroid-mediated renal mechanism.     

Atrial natriuretic factor in the pleural fluid of congestive heart failure patients

To investigate the possibility that atrial natriuretic factor might be secreted into the pleural fluid of patients with congestive heart failure who are known to have high concentrations of this new peptide hormone circulating in their plasma, six patients with class 2 New York Heart Association classified congestive heart failure had simultaneos measurement of plasma and pleural fluid atrial natriuretic factor concentrations. Atrial natriuretic factor was found in high concentrations in the pleural fluid of all of these patients. The concentration of atrial natriuretic factor in pleural fluid was nearly equal to the concentration in plasma of these patients. Their plasma levels were double the plasma concentration of this peptide hormone in 54 persons without congestive heart failure. These preliminary findings demonstrate that atrial natriuretic factor is present in pleural fluid of patients with congestive heart failure, but whether or not this secretion of atrial natriuretic factor into the pleural fluid helps the lung clear the fluid present in the lung in congestive heart failure cannot be determined from the present investigation.     

Serotonin and dopamine receptors in the rat pituitary gland: autoradiographic identification, characterization, and localization

S2 serotonin and D2 dopamine receptors were identified, characterized, and localized in rat pituitary gland by quantitative light microscopic autoradiography. [3H]Spiperone was used to localize S2 serotonin and D2 dopamine receptors. A high concentration of D2 dopamine receptors [1 microM 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (ADTN)- or sulpiride-displaceable [3H]spiperone binding] was found in the rat intermediate lobe with much lower concentrations present in the anterior and posterior lobes. Significant densities of cinanserin-displaceable [3H]spiperone binding sites (i.e. S2 serotonin receptors) were present in all three lobes of the pituitary gland. [125I]Lysergic acid ([125I]LSD) was used to characterize further and selectively visualize S2 serotonin receptors in the rat pituitary. Data analysis by densitometry showed that [125I]LSD binding the rat intermediate pituitary was saturable and of high affinity with an apparent dissociation constant (Kd) of 1.2 nM. Data from competition studies using a variety of compounds showed a S2 serotonin receptor profile at this [125I]LSD binding site in rat pituitary. The highest concentration of [125I]LSD binding sites was found in the intermediate lobe with progressively lower concentrations present in the posterior and anterior lobes, respectively. There is a uniform pattern of distribution of S2 serotonin and D2 dopamine receptors within each lobe of the rat pituitary gland. The results of the present study provide the first identification of S2 serotonin receptors in the pituitary and confirm the heterogeneous distribution of D2 dopamine receptors within the rat pituitary. These data provide further evidence for the importance of dopamine in regulating pituitary function and suggest a physiological role for serotonin in regulating pituitary hormone secretion.     

Age-related changes in lipid peroxidation products in rat adrenal gland

Chloroform-methanol extracts from rat adrenals at five different ages (2, 6, 12, 18 and 24 months), were studied by fluorescence. After obtaining excitation and emission spectra, fluorescence intensity was measured at 365 nm excitation and 455 emission for all time points of aging. An additional study of lipid peroxidation employing a thiobarbituric acid reaction was made. Fluorescence intensity increased during aging from 16.39 × 10³ arbitrary units of fluorescence per gram of tissue at 2 months, to 34.33 × 10³ units at 24 months. Thiobarbituric acid reaction products expressed in nmol of malondialdehyde per gram of adrenal increased from 172.97 at 2 months to 640.83 at 24 months. One way analysis of variance revealed a statistically significant difference (p<0.05 and p<0.01 respectively). The results show an age-related steady increase in lipid peroxidation products in rat adrenals and suggest their accumulation in lipofuscin granules.