Atrial natriuretic peptide, ANP(99-126), receptors in rat thymocytes and spleen cells

A single class of saturable, specific binding sites for the circulating form of atrial natriuretic peptides, ANP(99-126), was identified in rat thymus and spleen and in isolated thymocytes and spleen cells using quantitative autoradiographic techniques. In the thymus, the relative potency of ANP analogs to inhibit [125I] ANP(99-126) binding was ANP(99-126) = ANP(103-126) greater than ANP(111-126) greater than ANP(103-125). ANP(103-123) could not displace [125I]ANP(99-126) binding. Addition of ANP(99-126) stimulated the formation of cyclic GMP in isolated thymocytes and spleen cells in a dose-dependent manner. Our results indicate that immune cells have specific ANP receptors which could be coupled to guanylate cyclase activation and may play a role in the regulation of the immune response.     

Specific binding sites for prohormone atrial natriuretic peptides 1-30, 31-67 and 99-126

Two peptides with vasodilatory properties consisting of amino acids 1-30 and 31-67 of the 98 a.a. N-terminal end of the prohormone of atrial natriuretic factor (proANF) which circulates in man were investigated to determine if they have specific binding sites on membranes isolated from DDT1 MF-2 smooth muscle cells. Smooth muscle is a known biologic target of these peptides. Competitive binding experiments revealed that proANFs (1-30), (31-67), and (99-126) (i.e., C-terminus; ANF) each had specific and separate binding sites. The dissociation constants for proANFs (1-30), (31-67), and (99-126) binding were 0.11 nM, 4 nM, and 7.3 nM, respectively. The binding site concentrations for proANFs (1-30), (31-67), and ANF were 2.57, 59.91 and 40 fmols/10(6) cells, respectively. The number of binding sites per cell were 1548, 36,087, and 24,090, respectively, for proANFs (1-30), (31-67), and (99-126) (ANF). Each peptide bound to DDT1 MF-2 membranes between 10(-8) to 10(-11) M but could only bind to the other peptides' receptors at concentrations of 10(-6) and 10(-7)M. These results suggest that proANF(1-30) and proANF(31-67) do not work through the ANF receptor but rather have their own separate and distinct receptors that mediate their biologic effects.     

Dopaminergic mediation of the diuretic and natriuretic action of centrally administered rat atrial natriuretic factor (99-126)

Intracerebroventricular administration of either rat atrial natriuretic factor (99-126) or dopamine to conscious male hydrated rats resulted in an increase in urinaryvolume and sodium excretion. This activity was prevented, in both cases, by nonselective dopamine antagonist haloperidol (2.5 or 1.25 mg/kg sc, 18 and 2 hr before intracerebroventricular administration of atrial natriuretic factor). Our findings suggest that atrial natriuretic factor exerts its centrally mediated effects on sodium and water metabolism, at least in part, via a dopaminergic mechanism.     

Urodilatin (CDD/ANP-95-126) is not biologically inactivated by a peptidase from dog kidney cortex membranes in contrast to atrial natriuretic peptide/cardiodilatin (alpha-hANP/CDD-99-126)

Atrial natriuretic peptide (CDD/ANP-99-126) is rapidly inactivated by a membrane preparations from dog kidney cortex. Inactivation occurs by cleavage of the ring structure in the position between Cys-105 and Phe-106. A unique proteolytic product separated by HPLC on reverse-phase column appears as a single peak which elutes prior the intact peptide. In contrast, CDD/ANP-95-126 (urodilatin) which is released from the kidney is not destroyed by proteolysis using an identical membrane preparation.     

Natriuretic and diuretic action of centrally administered rat atrial natriuretic peptide (99-126): possible involvement of aldosterone and the sympathoadrenal system

Intracerebroventricular (ICV) administration of rat atrial natriuretic peptide (99-126) (rANP) to conscious male hydrated rats resulted in a dose-related increase in urinary volume and sodium excretion over a 6-h period of urine collection. A diminished mineralocorticoid effect on the kidneys may explain the natriuretic phenomenon. This hypothesis was tested by ICV rANP injection (1.25 microgram/5 microL) in conscious hydrated rats pretreated beforehand with d-aldosterone (20 micrograms/kg, ip). Although the absolute amount of sodium excreted was reduced, aldosterone did not affect rANP-induced sodium output at 1 and 3 h. Rats that were sham-operated or bilaterally adrenalectomized after 4 days were pretreated with aldosterone and given an oral water load followed by ICV rANP or saline. The possible participation of the peripheral sympathetic nervous system in the central action of rANP was evaluated in rats pretreated with 6-hydroxydopamine. In sympathectomized and adrenalectomized rats natriuresis and diuresis were still evident after rANP. Our results indicate that the natriuretic effect of ICV rANP is independent of mineralocorticoids. Likewise, diuresis and natriuresis can occur in the absence of the adrenal glands and are independent from the neural tone that the adrenergic system exerts on sodium reabsorption.     

Effects of adrenalectomy and aldosterone on the action of centrally administered rat atrial natriuretic peptide-(99-126)

Intracerebroventricular (i.v.t.) administration of rat atrial natriuretic peptide-(99-126) (rANP) increases urinary volume and sodium excretion, but the mechanism is undefined. A diminished mineralocorticoid effect on the kidneys may explain the natriuretic phenomenon. This hypothesis was tested by i.v.t. rANP injection (1.25 micrograms/5 microliters) in conscious, hydrated rats pretreated beforehand with d-aldosterone (20 micrograms/kg, i.p.). Although the absolute amount of sodium excreted was reduced, aldosterone did not affect rANP-induced sodium output at 1 and 3 h. Rats which were sham-operated or bilaterally adrenalectomized (ADX) after four days were pretreated with aldosterone and given an oral water load followed by i.v.t. rANP or saline. In ADX rats natriuresis and diuresis after rANP were still evident. Our results indicate that the natriuretic effect of i.v.t. rANP is unrelated to plasma levels of mineralocorticoids. Likewise, diuresis and natriuresis can occur in the absence of the adrenal glands.     

Phosphorylation and dephosphorylation of the natriuretic peptide urodilatin (CDD-/ANP-95-126) and the effect on biological activity

Urodilatin (CDD-/ANP-95-126), a new peptide hormone from human urine, is comprised of the same amino acid sequence as cardiodilatin (CDD-99-126/alpha-hANP) except for N-terminal extention by four amino acid residues. The presence of the recognition sequence Arg101-Arg-Ser-Ser104 for the cyclic AMP-dependent protein kinase enables rapid phosphorylation in the Ser104-position. Phosphorylation of urodilatin is associated with decreased vasorelaxant potency, while dephosphorylation of "phospho-urodilatin" by acidic phosphatase completely restores bioactivity.     

Specific binding sites for atrial natriuretic peptide (ANP) in cultured mesenchymal nonmyocardial cells from rat heart

Specific binding sites for atrial natriuretic peptide (ANP) were studied in cultured mesenchymal nonmyocardial cells (NMC) from rat heart. Binding study using 125I-labeled synthetic rat (r) ANP revealed the presence of a single class of high-affinity binding sites for rANP in cultured NMCs derived from both atria and ventricles; the apparent dissociation constant (Kd) was approximately 0.2 - 0.3 nM and the number of maximal binding sites was approximately 190,000 - 300,000 sites/cell. rANP significantly stimulated intracellular cGMP formation of cardiac NMCs in a dose-dependent manner (1.6 X 10(-8) M - 3.2 X 10(-7) M). rANP had no effect on synthesis of prostaglandin I2 by cultured cardiac NMCs. The physiological significance of ANP action on cardiac tissue remains to be determined.     

Immunoreactive atrial natriuretic peptide and autoradiographic distribution of atrial natriuretic peptide binding sites in the brain of the Antarctic fish, Chionodraco hamatus

The anatomical distribution of atrial natriuretic peptide (ANP)-immunoreactive structures and the autoradiographic localization of ANP binding sites were studied in the brain of the Antarctic fish, Chionodraco hamatus. ANP-containing elements were colocated with ANP binding sites in the dorsal medial and lateral subdivisions of the telencephalon, prethalamic nuclear complex, and in the nucleus of the medial longitudinal fasciculus of the mesencephalon. However, mismatching was observed in other brain regions, particularly at mesencephalic and metencephalic levels. In the pituitary, ANP immunoreactivity occurred only in the pars distalis, whereas ANP binding sites were localized in the whole pituitary. In this paper we describe the occurrence of ANP immunoreactivity and ANP binding sites in the brain and pituitary of an Antarctic fish. In particular, in the cerebellum and pituitary of C. hamatus, ANP binding sites are distributed in corresponding brain regions of dipnoans, amphibians and mammals. The immunocytochemical and histoautoradiographic data suggest that ANP acts as neuromodulator in the brain of C. hamatus. Moreover, the presence of ANP-like substances in tanycytes lining the diencephalic ventricle suggests a chemosensorial role for such liquor-contacting cells and a possible modulatory effect of ANP on the osmoregulation of the cerebrospinal fluid. Accepted: 3 April 2000     

Increased atrial natriuretic peptide (6-33) binding sites in the subfornical organ of water deprived and Brattleboro rats

Binding sites for rat atrial natriuretic peptide (6-33) (ANP) were quantitated in the subfornical organ of chronically dehydrated homozygous Brattleboro rats unable to synthesize vasopressin; heterozygous Brattleboro rats, their controls, Long Evans rats and Long Evans rats after 4 days of water deprivation. Brain sections were incubated in the presence of 125I-ANP and the results analyzed by autoradiography coupled to computerized microdensitometry and comparison to 125I-standards. Brattleboro rats and water deprived Long Evans rats presented a higher number of ANP binding sites than their normally hydrated controls. Our results suggest a role of ANP binding sites in the subfornical organ in the central regulation of fluid balance and vasopressin secretion.     

Localization of relaxin binding sites in the rat uterus and cervix by autoradiography

125I-labeled porcine relaxin was injected into 27-day-old rats treated with pregnant mare's serum gonadotropin (PMSG) and known target tissues for relaxin, the myometrium, endometrium and cervix, and putative control tissues, heart, thigh muscle and duodenum, examined for binding by autoradiography. Specific binding in the target tissues was demonstrated by simultaneous injection of excess unlabeled relaxin. Radioactivity was located and quantified by grain counts predominantly over the inner, circular muscle layer of the myometrium and the cervix and to a lesser extent over the outer longitudinal muscle layer of the myometrium and the endometrium. The route of injection, the circulation time, or counting grains in transverse or longitudinal sections of myometrium made little difference in these results. Ovariectomy decreased, but not significantly, the grain count in all of the target tissues studied and estrogen treatment of ovariectomized animals restored the numbers of grains to approximately that of intact PMSG-treated rats. The degree of binding of the cervix was approximately that of the circular myometrial muscle. This work confirms the presence of specific receptors for relaxin in the rat uterus and cervix of primed rats and it also suggests that the inhibitory action of relaxin upon the myometrium is primarily on the inner circular muscle layer.     

Regulation of atrial natriuretic factor-(99-126) secretion from neonatal rat primary atrial cultures by activators of protein kinases A and C

Atrial natriuretic factor (ANF) is stored in atrial myocytes as a prohormone (ANF-(1-126] and is cosecretionally processed to the circulating ANF-related peptides, ANF-(1-98) and ANF-(99-126). Recently, we have shown that the cosecretional processing of ANF can be replicated in primary cultures of neonatal rat atrial myocytes maintained under serum-free conditions and that glucocorticoids are responsible for supporting this processing activity. Activators of protein kinase C (phorbol esters and alpha-adrenergic agonists) and of protein kinase A (cAMP analogs, forskolin, and beta-adrenergic agonists) were tested for their abilities to alter the rate of ANF secretion from the primary cultures. ANF secretion was stimulated approximately 4-fold after a 1-h incubation of the cultures with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA); maximal release occurred at about 100 nM TPA. Reversed-phase high performance liquid chromatography analysis of secreted material indicated that the cells efficiently cosecretionally processed ANF under both basal and TPA-stimulated conditions. However, incubating the cultures for more than 1 h with TPA resulted in a blunted secretory response to further TPA challenge and a 40-50% decrease in the quantity of ANF in the cells. The alpha-adrenergic receptor agonist phenylephrine was also capable of stimulating ANF secretion by about 4-fold at a half-maximal dose of about 1 microM. Phenylephrine-stimulated ANF secretion was inhibited by the alpha 1-adrenergic antagonist prazosin with half-maximal inhibition occurring at approximately 1 nM. Forskolin, 8-bromoadenosine 3':5'-cyclic monophosphate, and N6-2(1)-O-dibutyryladenosine 3':5'-cyclic monophosphate inhibited basal, TPA- and phenylephrine-stimulated ANF secretion. The beta-adrenergic agonist isoproterenol partially inhibited phenylephrine-stimulated ANF secretion with the maximal effect occurring at 1 nM. These results indicate that ANF secretion from the neonatal rat atrial cultures is enhanced by activators of protein kinase C, and decreased by activators of protein kinase A, and that these secretory effects may be mediated through the actions of alpha- and beta-adrenergic receptors, respectively.     

Endopeptidase-24.11 in human plasma degrades atrial natriuretic factor (ANF) to ANF(99-105/106-126)

We previously demonstrated the presence of ANF(99-126), and ANF(99-126) cleaved between Cys105 and Phe106 (cleaved ANF), in human coronary sinus plasma. We now report that cleaved ANF is formed when synthetic ANF(99-126) is added to human plasma. When synthetic ANF(99-126) was incubated in heparinized human plasma, HPLC analysis showed two degradation products. The main product was shown by amino acid and sequence analysis to be cleaved ANF. Degradation of ANF was inhibited by EDTA and phosphoramidon. These findings are consistent with the action of endopeptidase EC 3.4.24.11, which may play an important part in the biological inactivation of ANF.     

Immunoreactive atrial natriuretic peptide in the guinea pig spleen

The presence of immunoreactive ANP precursor-like material in the guinea pig spleen is suggested. This is based on the following experimental evidence: An acidic extract of guinea pig spleen analysed by Sephadex G-50 gelfiltration contained 4.6 pmol/g wet tissue immunoreactive atrial natriuretic peptide (IR-ANP), coeluting with the 15 kDa synthetic ANP (2-126). Gelfiltrated IR-ANP material was further submitted to reverse phase high performance liquid chromatography and monitored by radioimmunoassay employing two antisera. One antiserum recognizes the C-terminal of ANP (1-126), the second is directed against the N-terminal sequence. Both antisera revealed material eluting with synthetic ANP (2-126). Furthermore, immunohistochemical analysis suggests this ANP-like material to be localized mainly at the periphery of the white pulp of the spleen. These findings link ANP with the immune system.     

The secretion of atrial natriuretic factor-(99-126) by cultured cardiac myocytes is regulated by glucocorticoids

Atrial natriuretic factor (ANF) is stored in mammalian atria primarily as ANF-(1-126), the precursor to the known circulating form of the hormone ANF-(99-126). When primary cultures of atrial myocytes were maintained in a complete serum-free medium, they contained and secreted an ANF-(1-126)-like peptide. The addition of dexamethasone to the culture medium, however, resulted in the secretion of a molecule with chromatographic characteristics identical to ANF-(99-126), although the intracellular storage form of ANF was unchanged. Radiosequencing and amino acid analysis confirmed that the cultures maintained in dexamethasone secreted authentic ANF-(99-126). Chronic exposure of the cells to dexamethasone also resulted in a significant increase in the quantity of immunoreactive ANF both contained and secreted by the cultures. Dexamethasone stimulated ANF processing and secretion by atrial cultures in a dose-dependent manner, with an approximate EC50 of 10 nM. This stimulation could be reversed by removing the glucocorticoid from the culture medium. ANF processing was also stimulated by the specific glucocorticoid receptor agonist RU 28362, and both DEX- and RU 28362-stimulated ANF processing was inhibited by the specific glucocorticoid receptor antagonist RU 38486. Ventricular cells, which possess few granules and release ANF in a constitutive fashion, were also capable of processing ANF in a glucocorticoid-dependent fashion. Medium freshly removed from atrial cultures did not convert ANF-(1-126) to ANF-(99-126) nor was exogenous ANF-(1-126) efficiently processed when added to the medium of actively processing cultures. These results indicate that the post-translational processing of ANF-(1-126) to ANF-(99-126) likely occurs within or in close association with the cardiac myocytes and is not dependent on the presence of large quantities of secretory granules. Furthermore, it is apparent that both the expression and the post-translational processing of ANF by cultured cardiac myocytes is specifically regulated by glucocorticoids.     

Atrial natriuretic peptide binding sites in the mammalian heart: Localization to endomural vessels

Summary The distribution of binding sites for atrial natriuretic peptide in cardiac ventricles of several mammalian species, including rat and human, was determined by in vitro autoradiography. The results revealed a unique anatomic localization of atrial natriuretic peptide binding sites to endomural vessels (Thebesian vessels), which communicate directly with the ventricular chambers. Digital image analysis indicated that these vascular channels possessed binding site densities comparable to those of the renal glomeruli a major target site for circulating atrial natriuretic peptide. In contrast, no specific labeling of branches of the coronary arteries and veins was detected. The discrete localization of atrial natriuretic peptide binding sites to this primitive cardiac circulatory system allows speculation as to the role of this hormone in the regulation of endocardialcirculation during cardiac development, normal ventricular function, and in coronary insufficiency.     

Localization of vasoactive intestinal peptide binding sites in the thymus and bursa of Fabricius of the chick

The purpose of this study was to determine the distribution of VIP binding sites in the thymus and bursa of Fabricius using receptor binding and autoradiographic techniques. Biochemical characterization of 125I-VIP binding sites determined two classes of specific binding sites in both tissues. The dissociation constants determined in the thymus were 1.12 nM and 88.5 nM, and in the bursa were 0.459 nM and 70.8 nM. Autoradiographic localization of 125I-VIP binding sites within the thymus demonstrated specific binding associated with the medullary region of the thymic lobule and the blood vessels in the interlobular and trabecular areas. Within the bursa of Fabricius, high densities of silver grains corresponded with vascular elements in the interfollicular regions, the epithelial border of the plicae, the muscular layer surrounding the organ, and the diffusely infiltrated area near the burso-cloacal duct.     

Localization and characterization of atrial natriuretic factor (ANF)-like peptide in the frog atrium

The distribution of ANF was studied in the heart of the frog (Rana ridibunda) using indirect immunofluorescence. ANF-like immunoreactivity was localized mainly in the right and left atrium, most of cardiocytes being intensively labelled. At the electron microscopic level, all secretory granules present in atrial cardiocytes contained ANF immunoreactive material. Using a specific radioimmunoassay, we found higher concentrations of ANF in the left atrium (208 +/- 25 ng/mg protein) than in the right atrium (120 +/- 16 ng/mg protein) whilst in the rat, the right atrium contains the highest ANF concentration. The concentration of ANF in the ventricle was 10 times lower than in the whole atrium (32 +/- 4 ng/mg protein). Sephadex G-50 gel filtration of atrial extracts showed that ANF-like immunoreactivity eluted in three peaks. Most of the immunoreactivity corresponded to high molecular weight material eluting at the void volume while 20% of the material co-eluted with synthetic (Arg 101-Tyr 126) ANF. These results indicate that frog cardiocytes synthetize a peptide which is immunologically and biochemically related to mammalian ANF.     

Visualisation of specific binding sites for atrial natriuretic peptide on non-neuronal cells of cultured rat sympathetic ganglia

Summary The distribution of atrial natriuretic peptide binding sites on cells in dissociated culture preparations of neonatal rat superior cervical ganglia and in explant cultures of rat thoracic sympathetic chain ganglia has been studied. The autoradiographic visualisation of atrial natriuretic peptide binding sites has been combined with the use of specific immunocytochemical markers for glial cells (antiserum to S-100 protein), fibroblasts (antiserum to fibronectin) and neurones (antiserum to protein gene product 9.5) in order to achieve unambiguous identification of the cell types in culture. Specific binding sites for rat¹²⁵I-atrial natriuretic peptide_(1–28) were observed over subpopulations of fibronectin-like-immunoreactive fibroblasts and S-100-like-immunoreactive glia in the dissociated superior cervical ganglion cultures. However, only a subpopulation of fibronectin-like-immunoreactive fibroblasts possessed atrial natriuretic peptide binding sites in the explant culture preparations. No atrial natriuretic peptide-like-immunoreactive cells were present in either culture. The distribution of autoradiographic grains over individual cell surfaces in culture was uniform, but there were distinct differences in the density of labelling of single cells of the same type. This apparent variation in the number of binding sites on glial cells and fibroblasts in culture did not seem to be related to the morphology of the cells or the surrounding cell types. No sympathetic neurones were labelled with autoradiographic grains in either the dissociated or explant culture preparations. However, the presence of atrial natriuretic peptide binding sites on non-neuronal cells of sympathetic ganglia in culture may be linked to the relationship between atrial natriuretic peptide and the sympathetic nervous system.     

High- and low-affinity binding sites for vasoactive intestinal peptide (VIP) in the rat kidney revealed by light microscopic autoradiography

The distribution of VIP binding sites in rat kidney and adrenal gland has been examined by light microscopic autoradiography. A fully characterized mono-iodinated molecular form of VIP (M-125-I-VIP) which maintains the biological activity of the native peptide, was used for this study. Two types of VIP binding sites, with high and low affinity, have been identified. High affinity sites are associated with (i) glomerular structures in the cortex, (ii) the inner stripe of the outer medulla, possibly corresponding to Henle's loops and distal tubules, (iii) radiated structures in the inner zone of the medulla, likely to represent labeling of collecting ducts and/or vascular bundles and (iv) the adrenal cortex. Autoradiographic grains associated with low affinity sites are present diffusely throughout the renal cortex, possibly corresponding to labeling of tubular and/or vascular structures, and throughout the adrenal gland. These observations further delineate a role of VIP in renal and neuroendocrine function.