Atrial natriuretic peptide in lymphoid organs of various species

1. Evidence for the occurrence of atrial natriuretic peptide (ANP) in various lymphoid organs of different species (rat, mouse, pig, chicken) is provided. 2. ANP precursor material (1-126) as well the physiologically active ANP (99-126), were identified by chromatographic analysis and RIA in extracts of thymus, spleen and lymph nodes of rat, mouse and pig. 3. mRNA coding for ANP was demonstrated both in the thymus and in isolated thymocytes of these species. Furthermore, mRNA for ANP was detected in spleen and lymph nodes (rat and pig). 4. The bursa of Fabricius, thymus glands and spleen of chickens were also shown to express mRNA coding for ANP. 5. These findings provide a firm basis for a link of ANP to the immune system, a novel aspect of possible biological functions of this peptide.     

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.     

The rat thymus--a site of atrial natriuretic peptide synthesis

The atrium of the heart has been demonstrated to represent the major site of synthesis of atrial natriuretic peptide (ANP), a potent natriuretic, diuretic and vasoactive hormone. Our recent studies revealed ANP-like material outside the heart, namely, in lymphoid follicles of the intestine and in the thymus, and now we report data demonstrating the thymus as a site of synthesis for ANP. The experimental evidence is as follows: firstly, the immunoreactive material detected corresponds chromatographically with the precursor of ANP. Secondly, the thymus contains mRNA for ANP. Thirdly, immunohistochemistry locates ANP-like material to cortical thymocytes with particularly dense staining in the subcapsular areas of the thymus. Interestingly, both ANP-like material and the mRNA coding for ANP were expressed to a larger extent in newborn rats as compared to adult animals, suggesting that ANP may be involved in the development and/or function of T-cells.     

Atrial natriuretic peptide inhibits nitric oxide synthesis in mouse macrophages

The atrial natriuretic peptide (ANP) affects cardiovascular physiology, and, as has been suggested more recently, exerts immunomodulatory activities. In this context, we examined the effect of ANP on nitric oxide (NO) synthesis in murine bone marrow derived macrophages as well as in peritoneal macrophages. Cultured macrophages were stimulated with lipopolysaccharides (LPS 0.1–10 μg/ml) and NO synthesis was monitored by measuring increased concentrations of NO₂ in the medium. In initial experiments employment of N^G-monomethyl-L-arginine (L-NMMA) and dexamethasone, two specific inhibitors of nitric oxide synthase (NOS), confirmed the presence of inducible NOS activity in the cells. Exposure of cells to rat ANP99–126 in the range of 10⁻⁸ to 10⁻⁶ M significantly decreased LPS induced NO synthesis over 24 hours of incubation. Thus, ANP may alter macrophage function by affecting their nitric oxide synthesizing pathway.     

The use of a monoclonal antibody to measure plasma atriopeptins in rat

A monoclonal antibody with specificity for atrial natriuretic peptides (ANP) was produced, that can be used for the radioimmunological determination of ANP-immunoreactivity (ANP-IR) in rat plasma. The antibody recognizes atriopeptin I, II, III, as well as alpha-hANP and alpha-hANP fragment (7-28) and does not crossreact with ANP-fragments (13-28) and (18-28). Plasma levels of ANP-IR in conscious Wistar rats were determined before and after volume-loading. Basal plasma levels of ANP-IR were 108 +/- 12 pg/ml, and after volume-loading increased to 800 +/- 59 pg/ml.     

Lower number of atrial natriuretic peptide receptors in thymocytes and spleen cells of spontaneously hypertensive rats

Spontaneously hypertensive rats (SHR) have a much lower number of atrial natriuretic peptide (ANP) receptors in thymus and spleen from young and adult animals than age-matched normotensive controls. In spite of this low receptor concentration, the ANP-stimulated cyclic GMP response in isolated thymocytes and spleen cells from SHR was similar to that of normotensive control rats. Alterations in ANP receptor concentration in thymus and spleen of SHR may be related to the immune abnormalities described in these animals, and to the pathophysiology of genetic hypertension.     

Localization of atrial natriuretic peptide, ANP-(99-126) binding sites in the rat thymus and spleen with quantitative autoradiography

Binding sites for atrial natriuretic peptide, ANP-(99-126) were studied in lymphoid organs of the rat with quantitative autoradiography. Tissue sections were incubated in the presence of 0.13 nM 125I-ANP-(99-126) followed by autoradiography using [3H]-Ultrofilm, and the results were analyzed by computerized densitometry and comparison to 125I-standards. Specific ANP binding sites were localized in the medulla and the cortex of the rat thymus and in the white pulp of the rat spleen, with apparent binding sites concentrations of 93, 65, and 126 fmol/mg protein, respectively. The presence of ANP binding sites in areas related to the maturation and function of lymphocytes, and to the production of thymic hormones, suggests the possibility of a role of circulating ANP in the modulation of the immune response.     

Infradian Variation of Rat Thymic and Splenic mRNA to Novikoff’s Hepatoma

During the immune response to rat tumor cells there is participation of the thymus and the spleen via the synthesis of antibodies and immune cellular elements. During this process different mRNAs of both organs are synthesized. Here is presented the infradian variation of mRNA synthesis of immunized rat thymus and spleen to Novikoff’s Hepatoma (NH) cells. These cells were maintained and transferred in ascitic manner in the peritoneal cavity of Sprague Dawley male rats standardized with 12 hours (h) of light and 12 h of darkness. Aliquots of 1 x 10 6 NH dead cells were innoculated intraperitoneally into rats after being exposed during 30 minutes to ultraviolet light and incubated at 37°C with 50 U of neuraminidase/ml during 70 minutes. Groups of 3 controls and 3 immunized rats were killed at the same circadian timepoint (10 a.m.) under anesthesia every 3 days during a span of 18 days. At each time spleens and thymus of each group were harvested and pooled in order to isolate their mRNA. Isolation of the rRNA and mRNA of control and immunized rats was performed by affinity chromatography employing oligothymidylic acid cellulose columns. Analysis of variance demonstrated a significant (p <0.03) higher synthesis of immune (i)mRNA of rat spleen, starting on 3 days after immunization with NH cells and reaching the higher levels on 6 and 18 days after immunization and lower levels on 9, 12, 15 days after immunization. Same effect is also observed in the synthesis of imRNA rat thymus 6 days after immunization, however, there was not difference with the intact rat thymus mRNA on 3, 9 and 12 days after immunization. Interestingly, there was observed an increased synthesis of intact rat thymus mRNA 15 days after inoculation. During this cyclic synthesis of thymic and spleenic imRNAs it seems that the spleen plays the role of a possible pacemaker of the coordinated immune response to NH cells.     

Role of natriuretic peptide signaling in modulating asthma and inflammation

Atrial natriuretic peptide (ANP), the C-terminal peptide comprising residues 99-126 of the pro-ANP hormone, has been studied for 3 decades for its cardiovascular effects. Recent reports suggest that it plays a significant role in modulation of the immune system. Immune cells, including macrophages, dendritic cells, and T lymphocytes, express receptors for ANP. ANP plays a significant role in shaping the early immune response to environmental antigens and may play a critical role in the interaction between cells of the innate and adaptive immune systems; it also appears to be involved in polarizing the immune response to allergens. Thus, ability to alter the magnitude of natriuretic peptide receptor A (NPRA) signaling could be exploited to develop therapeutics for several allergic diseases, including asthma. This report will review and critically evaluate the role of the ANP pathway in asthma and inflammation.     

Immunohistochemical localization of atrial natriuretic peptide in the developing and adult mammalian kidney

The discovery, within the last decade, of atrial natriuretic peptide (ANP), a family of peptides with natriuretic/diuretic and vasorelaxant properties, has prompted much research into the mechanisms and sites of action of ANP within the kidney. In the present study, ANP was localized in the kidneys of several mammalian species by immunohistochemical techniques 1) to identify possible sites of synthesis; 2) to compare the localization of ANP to known physiological effects; 3) to determine species differences, if any, in ANP localization; and 4) to study the development of ANP immunoreactivity in the fetal and neonatal rat kidney. Using an antibody against rat ANP, IV, ANP was localized exclusively on the proximal convoluted tubule (PCT) brush border and within intercalated cells of the outer medullary and cortical collecting tubules and ducts of adult mouse, rat, pig, monkey, and human kidneys. The development of ANP immunoreactivity paralleled the differentiation and maturation of collecting duct epithelium in rat fetal kidney. Atrial natriuretic peptide found within intercalated cells of the cortical and outer medullary collecting ducts may be the result of endogenous synthesis and, following secretion, may be available to receptors in the inner medullary collecting ducts.     

Ontogenesis of rat immune system: Proteasome expression in different cell populations of the developing thymus

Immune proteasomes in thymus are involved in processing of self-antigens, which are presented by MHC class I molecules for rejection of autoreactive thymocytes in adults and probably in perinatal rats. The distribution of immune proteasome subunits LMP7 and LMP2 in thymic cells have been investigated during rat perinatal ontogenesis. Double immunofluorescent labeling revealed LMP7 and LMP2 in thymic epithelial and dendritic cells, as well as in CD68 positive cells - macrophages, monocytes - at all developmental stages. LMP2 and LMP7 were also detected by flow cytometry in almost all thymic CD90 lymphocytes through pre- and postnatal ontogenesis. Our results demonstrate that the immune proteasomes are expressed in all types of thymic antigen presenting cells during perinatal ontogenesis, suggesting the establishment of the negative selection in the thymus at the end of fetal life. The observation of the immune proteasome expression in T lymphocytes suggests their role in thymocyte differentiation besides antigen processing in thymus.     

An immunocytochemical survey of endocrine cells in the gastrointestinal tract of chicks at hatching

Summary The ultrastructure of the micro-environment of the fully functional rat thymus was studied. The thymus consists of two discrete compartments, viz., an epithelial and a mesenchymal compartment. Thymus fibroblasts/fibrocytes, mast cells and granulocytes, are restricted to the mesenchymal compartment. The thymocyte maturation process seems to occur in the epithelial compartment in a network of reticular epithelial cells. The cortex is finely meshed and filled with proliferating thymocytes and some scattered macrophages. Moreover, in the medulla vacuolated epithelial cells form part of a loosely meshed reticulum which is filled with thymocytes and interdigitating cells (IDCs). IDCs frequently contain Birbeck granules and appear to be phagocytic. Together with macrophages, they probably enter the thymus, predominantly in the cortico-medullary region, and cross the separating wall between the two compartments. Some functional aspects of the non-lymphoid cells and in particular the IDCs, which form the micro-environment of the thymus, are discussed with respect to T-cell development.     

Evidence for the presence of ANP-precursor material in the rat thymus

Acidic extraction of the thymus from two day old rats followed by purification on Sephadex G-50 gelfiltration revealed the presence of atrial natriuretic peptide-like material (IR-ANP) by radioimmunoassay. Verification of the obtained immunoreactivity has been achieved by the use of two different types of antisera, i.e. two antisera directed against ANP (99-126), the other antiserum recognizing the N-terminal fragment (11-37) of the precursor ANP (1-126) molecule. In addition, reverse phase high performance liquid chromatography (RP-HPLC) and high performance gel permeation chromatography (HP-GPC) monitored by the three antisera have been employed for analysis of the extracted IR-ANP. In both systems the IR-ANP corresponded to the 15 kDa-ANP molecule (1-126). Furthermore, by using immunohistochemical techniques a distinct localization of the IR-ANP material could be demonstrated. The outer cortical area of the thymus, containing mostly lymphoid cells, showed extensive immunostaining with the three different antisera. The data reported here indicate that the rat thymus is a source of ANP.     

The thymus as a target for mycobacterial infections

Mycobacterial infections are among the major health threats worldwide. Ability to fight these infections depends on the host's immune response, particularly on macrophages and T lymphocytes produced by the thymus. Using the mouse as a model, and two different routes of infection (aerogenic or intravenous), we show that the thymus is consistently colonized by Mycobacterium tuberculosis, Mycobacterium avium or Mycobacterium bovis BCG. When compared to organs such as the liver and spleen, the bacterial load reaches a plateau at later time-points after infection. Moreover, in contrast with organs such as the spleen and the lung no granuloma were found in the thymus of mice infected with M. tuberculosis or M. avium. Since T cell differentiation depends, to a large extent, on the antigens encountered within the thymus, infection of this organ might alter the host's immune response to infection. Therefore, from now on, the thymus should be considered in studies addressing the immune response to mycobacterial infection.     

Macrophage regulation of DNA synthesis in lymphoid cells: effects of a soluble factor from macrophages

Addition of rat peritoneal macrophages to nonadherent rat spleen cells in culture results in enhancement or suppression of DNA synthesis depending on the ratio of macrophages to lymphocytes. At high ratios of macrophages to lymphocytes (1:5), suppression can be observed as early as four hours. Macrophages suppress incorporation of thymidine (TdR) by nonadherent spleen, thymus and bone marrow cells, in most instances, to less than 5% of that observed in culture to which macrophages were not added. In the presence of macrophages, incorporation of [³H]uridine and [¹⁴C] amino acids by spleen cells was also moderately suppressed. Based on ⁵¹Chromium release and dye exclusion assays, it appears that suppression is not due to cytotoxicity. Furthermore, suppression of [³H]TdR incorporation by nonadherent spleen cells is reversible, in the presence of an antigenic stimulus, following removal of the macrophages from the cultures. The suppressive effects are not elicited by extracts of macrophages, freeze-thawed or heated macrophages, but appear to be due to a low molecular weight, heat stable factor released into the macrophage culture fluid.     

Distribution patterns and coexistence of neurohormonal peptides (ANP,BNP, NPY,SP, CGRP,enkephalins) in chromaffin cells and nerve fibers of the anuran adrenal organ

Summary Immunohistochemical techniques were used to study the adrenal organs of the anuran species Rana esculenta, Caldula pulchra and Bufo marinus with respect to the distribution and coexistence of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), substance P (SP), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), Leu-enkephalin (Leu-ENK). Met-enkephalin-Arg-Phe (MEAP) and dynorphin A 1–17 (DYN). Antisera against enzymes involved in catecholamine synthesis, i.e., dopamine--hydroxylase (DBH) and tyrosine hydroxylase (TH), were used for the identification of chromaffin cells. ANP-immunoreactive (-IR) cells occurred in high densities (30%–70% of the total cell population) in all species investigated. In C. pulchra and B. marinus, BNP-IR cells constituted a population of non-DBH-IR and non-TH-IR cells that were different from the ANP-IR cells. A large proportion of the adrenal cells (10%–55%) were immunoreactive to Leu-ENK, and a minority (2%–5%) showed MEAP-immunoreactivity. DYN-immunoreactivity was not observed. The anurans studied exhibited small numbers of SP-IR, CGRP-IR and NPY-IR cells. Immunoreactivities for ANP+Leu-ENK and Leu-ENK+ MEAP were shown to coexist. In C. pulchra and B. marinus, immunoreactions for ANP+NPY, ANP+SP and SP+CGRP were also colocalized. Except for DYN, all neurohormonal peptides also occurred in intra-adrenal nerve fibers. SP-IR fibers also displayed CGRP-immunoreactivity and some Leu-ENK-IR fibers contained MEAP-immunoreactivity. In C. pulchra, NPY-IR fibers were found that also showed ANP-immunoreactivity.Some results of this investigation have been presented in abstract form (Reinecke et al. 1991).     

Discovery of a natriuretic peptide family and their clinical application

The identification of atrial natriuretic peptide (ANP) induced an explosive series of studies on the new peptide involved in control of the circulation, both in the basic and clinical fields. During the first decade of ANP research surprising progress has been made, revealing that the heart is an endocrine organ regulating the circulation system. ANP has been developed as a diagnostic tool and as a therapeutic drug for cardiac failure. In the second decade, brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) were identified, unveiling new profiles of this peptide family. Although BNP is also a circulating hormone that shares a common receptor with ANP, it is different from ANP in its' synthesis and secretion. Plasma concentration of BNP reflects the severity of heart failure in patients in a dramatic fashion, much moreso than ANP. Thus, BNP has been developed as a powerful diagnostic tool for cardiovascular diseases. The third congener, CNP, having a receptor of its own, was initially thought to function only in the brain. CNP was subsequently found to be produced from vascular endothelial cells and macrophages, indicating that CNP is a local regulator and also an antiproliferative factor in the vascular cell system, rather than a circulating hormone. Trials for the clinical application of CNP have also been discussed.     

Abundant expression of thromboxane synthase in rat macrophages

The cloned cDNA for rat thromboxane (TX) synthase with a size of 1851 bp contained a 1599-bp open reading frame which encoded a 533-amino acid protein sharing 79.7% identity with human TX synthase. RNA blot analysis was carried out with rat cells and tissues. Rat peritoneal macrophages most abundantly expressed mRNA for TX synthase, and its level was not changed by in vivo stimulation of casein. Bone marrow, spleen, lung and thymus also expressed the TX synthase gene. These findings suggest the possibility that TXA₂ plays a role in the immune system.     

Molecular cloning and characterization of a novel human gene (ANP32E alias LANPL) from human fetal brain

Leucine-rich acidic nuclear protein (LANP) is a member of the leucine-rich repeats (LRRs) superfamily. Here we report on a human homologue of LANP, encoded by the gene ANP32E (alias LANPL). The gene was cloned and identified during large-scale sequencing analysis of a human fetal brain cDNA library. The human protein shared 70% amino acid identity with rat LANP. According to bioinformatics analysis, ANP32E is located on chromosome 1q22. RT-PCR analysis indicates that ANP32E was expressed in human peripheral blood leukocytes, colon, small intestine, prostate, thymus, spleen, skeletal muscle, liver and kidney.