Lack of atrial natriuretic peptide receptors in human aldosteronoma

The effect of synthetic alpha-human atrial natriuretic peptide (ANP) on aldosterone secretion was studied in human aldosterone producing adrenocortical adenoma obtained surgically from a patient with primary aldosteronism and in human apparently normal adjacent adrenal cortical tissues obtained from a patient with pheochromocytoma, in vitro. Apparently normal adrenal cortical tissue responded to ANP with the known inhibition of aldosterone secretion. In contrast, the aldosterone producing adenoma did not respond to ANP. When stimulated by either ACTH or angiotensin II, there is no inhibition by ANP in the adenoma tissue, whereas normal tissue was inhibited. Immunohistochemical examination utilizing an ANP-receptor antiserum demonstrated that there was no evidence of binding site in the cortical adenoma, in contrast, zona glomerulosa cells in the cortical tissues adjacent to either aldosterone producing adenoma or pheochromocytoma were densely stained. This apparent lack of ANP-receptors is an associated finding with the hypersecretion of aldosterone in the aldosterone producing adenoma.     

Presence of functional receptors for atrial natriuretic peptide in human pheochromocytoma

Pheochromocytoma, a catecholamine-secreting adrenomedullary tumor, has been shown to contain the functional receptor for human atrial natriuretic peptide(h-ANP). Release of catecholamines from tissue slices of pheochromocytoma was inhibited by h-ANP in a dose-dependent manner. Binding assays using 125I-ANP revealed a single class of high affinity binding sites for ANP. When covalently tagged with 125I-ANP and electrophoresed under non-reducing and reducing conditions, the receptor migrated as a 140-kDa band and a 70-kDa band, respectively, reflecting its disulfide-linked subunit structure. The presence of ANP receptor in pheochromocytoma was further demonstrated by immunohistochemistry; the tumor was positively stained with an antireceptor antiserum. The antiserum was also useful to establish the zona glomerulosa localization of ANP receptor in the normal human adrenal gland.     

Human pheochromocytoma cells studied in culture contain large amounts of DSIP-like material.

Delta sleep-inducing peptide (DSIP)-like immunoreactive (LI) material has been detected in nine different human pheochromocytoma tumors by immunocytochemistry. In primary tumors subjected to indirect immunofluorescence a variable number of tumor cells (25-75%) showed positive cytoplasmic labeling after incubation with DSIP antiserum. Tumor cells grown in culture were strongly labeled by the DSIP antiserum with DSIP-LI concentrated to cell bodies. Electron microscopic immunocytochemistry (immunogold labeling) of pheochromocytoma cells demonstrated DSIP-LI over the dense core of secretory granules. The presence of DSIP-LI in several HPLC fractions from conditioned culture media indicates secretion of DSIP-LI from cultured pheochromocytoma cells. The observations suggest that DSIP-LI is synthesized and stored in secretory granules before release. The different HPLC profiles from each of the tumors may reflect differences in processing or turnover of DSIP-LI in pheochromocytoma cells.     

Regulated expression of atrial natriuretic peptide-like immunoreactivity in cultured bovine adrenomedullary chromaffin cells

We show that cultured bovine adrenal chromaffin cells can be stimulated to produce atrial natriuretic peptide-like immunoreactivity (ANP). ANP levels increased 5-fold in response to either forskolin or phorbol ester treatment, and 17-fold after depolarization by 40 mM potassium. These agents appear to act through distinct second messenger mediated pathways to cause increased accumulation of ANP. When forskolin and phorbol ester were added simultaneously, their effects were synergistic: ANP levels increased 30-fold, more than the product of the increases achieved by treatment with either drug alone. The ANP present in bovine chromaffin cell extracts appeared immunologically identical to human ANP(1–28). By gel filtration, the immunoreactive ANP extracted from chromaffin cells was in a high molecular weight form, although HPLC fractionation revealed that a portion of the total immunoreactivity could be eluted with the same retention time as synthetic ANP standards.     

Immunoreactive atrial natriuretic polypeptides in the adrenal medulla and sympathetic ganglia

Atrial natriuretic polypeptide (ANP)-like immunoreactivity was found in the rat adrenal gland by using indirect immunofluorescence and peroxidase-antiperoxidase techniques. ANP-like immunostaining was present in most of chromaffin cells with varying degrees of immunoreactivity. The majority of medullary cells displayed very intense immunostaining, and several clusters revealed weaker immunostaining. No staining was found in the adrenal cortex or in the nerve fibers in this organ. In the consecutive sections treated for dopamine-beta-hydroxylase (DBH), apparently all medullary cells had intense immunofluorescence for DBH and its distribution pattern was very similar to that for ANP-like immunoreactivity. While phenylethanolamine N-methyltransferase (PNMT) immunoreactive cells largely corresponded to the intensely stained ANP-like immunoreactive cells, suggesting that adrenaline cells contained a large amount of ANP-like substance, noradrenaline cells contained a smaller amount of this substance than adrenaline cells. Ultrastructural study showed that end-products due to the immunoreaction with the ANP antiserum were primarily associating with chromaffin granules. In addition, the presence of ANP-like immunoreactivity was investigated in several sympathetic ganglia of the rat. No principal ganglion cells were ANP-positive, whereas a few small intensely fluorescent (SIF) cells were ANP-immunoreactive. The present findings suggest that catecholamines coexist with ANP which has a natriuretic and vasodilating effect, in adrenal medullary cells and SIF cells in several rat sympathetic ganglia, but not in principal ganglion cells.     

Different patterns of expression of five neuropeptides in the adrenal gland and kidney of two species of frog

The aim of this study was to demonstrate in the adrenocortical and renal tissues of two species of frog, Rana italica and Rana esculenta, the presence and distribution of five neuropeptides: atrial natriuretic peptide (ANP), Leu-enkephalin (Leu-ENK), neuropeptide Y (NPY), substance P (SP) and vasoactive intestinal peptide (VIP).In anurans, the adrenal medulla is the site for the synthesis, storage and secretion of not only catecholamines but also various peptides. These peptides should not be regarded only as neurotransmitters or modulators for the secretion of catecholamines, but also as hormonal substances that induce systemic effects.All the peptides studied (ANP, Leu-ENK, NPY, SP and VIP) are present in both organs. However, different patterns of expression were observed for some of the peptides in two frogs.Immunopositivity to ANP was found in small clusters of chromaffin cells in both frogs whereas a clear strong positivity was present only in Rana esculenta kidney. Large clusters of chromaffin cells were immunoreactive to Leu-ENK in Rana italica but there were approximately 25% fewer compared to the positive cells present in Rana esculenta. Epithelial cells of renal tubules showed strong immunopositivity to Leu-ENK in Rana esculenta but not in Rana italica. A large number of adrenal cells (70–80%) were immunoreactive to NPY in Rana italica, while in Rana esculenta this peptide was localized in small clusters of chromaffin cells. Both frogs showed many NPY-positive cells in kidney. Many chromaffin cells were found positive to SP and VIP. A strong positivity was also observed in kidney in both frogs. These observations suggest a possible role of these peptides in the control of the physiological functions of adrenal glands and kidney of the two species of frogs studied.     

Release of neuropeptide Y from pheochromocytomas

To investigate the release of neuropeptide Y (NPY) from the pheochromocytomas, we studied the relationship between the plasma and tumor tissue immunoreactive (IR) NPY concentrations in 13 patients with pheochromocytoma and measured the IR-NPY concentration in plasma samples obtained by catheter from several veins (jugular veins, superior vena cava, renal veins, adrenal veins and inferior vena cava) in 2 patients with pheochromocytoma. The plasma IR-NPY concentration in 13 patients with pheochromocytoma ranged from 118 to 1460 pg/ml and the concentration in 10 of 13 patients with pheochromocytoma was above 290 pg/ml (the upper limit of normal range). The tumor tissue IR-NPY ranged from 0.025 to 95.3 micrograms/g wet tissue. Plasma IR-NPY was parallel with tumor tissue IR-NPY in 13 cases of pheochromocytoma (r = 0.76, P less than 0.01). The highest concentration of IR-NPY was found in plasma obtained from the drainage vein from a tumor among the plasma samples obtained from several veins in 2 cases of pheochromocytoma. These findings indicate that in patients with pheochromocytoma, NPY is in most cases excessively released from the tumors into the systemic circulation and plasma IR-NPY in the periphery is increased.     

Immunohistochemistry and immunocytochemistry of atrial natriuretic polypeptide in porcine heart

Specific granules in porcine hearts were observed in atrial cardiocytes, Purkinje fibers, and transitional cells of the ventricle. These granule-containing cells were immunohistochemically stained by applying the avidin-biotin-peroxidase complex method using an antiserum against alpha-human atrial natriuretic polypeptide (ANP). Immunoelectron microscopy of sections stained using the immunogold method indicated that these specific granules are storage sites of ANP. Furthermore, an impulse-conducting system consisting of immunoreactive cells was clearly distinguishable from nonimmunoreactive ventricular cardiocytes. We conclude that specific-granule-containing cells, i.e., ANP-producing cells, are located in both the atrial walls and the ventricular impulse-conducting system. The presence of ANP may be correlated with impulse conduction.     

Amidated joining peptide in the human pituitary, gut, adrenal gland and bronchial carcinoids. Immunocytochemical and immunochemical evidence

The distribution of the proopiomelanocortin-derivated amidated joining peptide (JP-N) was examined in the human pituitary gland, adrenal gland, gut and in three bronchial carcinoids. Double immunostaining showed coexistence of immunoreactive JP-N and other proopiomelanocortin derivatives, e.g., ACTH, beta-endorphin, Pro-tau-MSH, in the pituitary gland and adrenal medulla. The JP-N immunoreactive cells in the adrenal medulla were identified as a subpopulation of adrenaline-producing cells by means of an antiserum against phenylethanolamine N-methyltransferase. In the gut immunoreactive JP-N was costored with somatostatin in endocrine cells. Using radioimmunoassay, JP-N was found in higher concentrations than ACTH and alpha-MSH in the gut but not in the adrenal gland. Gel chromatography of gastric antrum and adrenal gland extracts showed three and two dominating components of immunoreactive JP-N, respectively, but under reduced conditions most of the immunoreactive material appeared as of low molecular weight in both extracts. In conclusion, immunoreactive JP-N is a major product from the processing of proopiomelanocortin in human extrapituitary tissues. The molecular forms of immunoreactive JP-N correspond to previous findings in the human pituitary gland.     

Localization and characterization of evolutionarily conserved chromogranin A-derived peptides in the rat and human pituitary and adrenal glands

Chromogranin A (CgA) is a neuroendocrine protein that undergoes proteolytic cleavage in secretory granules. The aim of the present study was to characterize the peptides WE14 and EL35 that are derived from evolutionarily conserved regions of CgA in rat and human endocrine tissues. In the rat pituitary, HPLC analysis revealed that WE14 is present as a single immunoreactive peak, whereas EL35 elutes in two molecular forms. Authentic WE14 is also produced in both rat and human adrenal glands, while EL35 displays a variable elution profile depending on the tissue extract, indicating the existence of different forms of EL35 in these tissues. Immunohistochemical labeling of the rat pituitary showed that WE14 and EL35 occur in gonadotropes and melanotropes, but not in corticotropes. A strong immunoreaction for both peptides was also observed in rat adrenochromaffin cells. In the human adrenal gland, the WE14 and EL35 antisera revealed intense labeling of adrenomedullary cells in adult and nests of chromaffin progenitor cells in fetal adrenal. Finally, WE14 and EL35 immunoreactivity was detected in pheochromocytoma tissue where WE14 occurred as a single immunoreactive form, while EL35 displayed different forms. The observations that WE14 and EL35: (1). have been preserved during vertebrate evolution, (2). are processed in a cell-specific manner, and (3). occur during ontogenesis of the adrenal gland strongly suggest that these peptides play a role in endocrine tissues. In addition, the existence of differentially processed CgA-derived peptides in normal and tumorous tissues may provide new tools for the diagnosis and prognosis of neuroendocrine tumors.     

Carbachol-Induced Cosecretion of Immunoreactive Atrial Natriuretic Peptides with Catecholamines from Cultured Bovine Adrenal Medullary Cells

The distribution and secretion of atrial natriuretic peptides (ANPs) were investigated in bovine adrenal medulla. (1) Cultured bovine adrenal medullary cells (2 x 10(6)/dish) contained 100.4 +/- 6.0 fmol of immunoreactive ANP (IR-ANP) and 207.3 +/- 6.6 nmol of catecholamines as epinephrine plus norepinephrine. (2) Stimulation of nicotinic but not muscarinic acetylcholine receptors caused a cosecretion of IR-ANP and catecholamines corresponding to the ratio of IR-ANP to catecholamines in cultured bovine adrenal medullary cells. (3) Carbachol-stimulated secretion of IR-ANP was dependent on the presence of extracellular Ca2+. (4) Chromaffin granules isolated from bovine adrenal medulla contained large amounts of IR-ANP and catecholamines, in the same ratio as did cultured adrenal medullary cells. (5) Reverse-phase HPLC analysis showed that both stored and secreted IR-ANP consisted of two components, which eluted at the position of ANP(99-126) or ANP(1-126). These results indicate that ANPs are stored as ANP(99-126) and ANP(1-126) in chromaffin granules, and are cosecreted in parallel with catecholamines in a Ca2+-dependent manner by the stimulation of nicotinic acetylcholine receptors.     

Spontaneous neurite outgrowth and vasoactive intestinal peptide-Iike immunoreactivity of cultures of human paraganglioma cells from the glomus jugulare

Summary The chief cells of paraganglionic tissues have morphological and functional similarities to adrenal chromaffin cells, and both cell types are derived from the neural crest. In the present investigation cells from two glomus jugulare paragangliomas were studied in culture. Approximately 50% of the cells from one tumor, and 7% from the other spontaneously formed neurite-like processes. Numerous granular and agranular synaptic-like vesicles also appeared in the process-forming cells. In contrast to findings with normal and neoplastic adrenal chromaffin cells, addition of nerve growth factor (NGF) to the culture medium had no major effects on proportion of cells with processes. Dexamethasone caused only a small decrease in process length. Culturing of the tumors also appeared to promote production of material with VIP-like immunoreactivity. It is concluded that the phenotype of paraganglioma as well as pheochromocytoma cells may be altered in vitro. Responsiveness to specific factors such as NGF or steroids, however, may vary for related tumor cell types in different anatomic locations.     

Immunohistochemistry and immunocytochemistry of atrial natriuretic polypeptide in porcine heart

Summary Specific granules in porcine hearts were observed in atrial cardiocytes, Purkinje fibers, and transitional cells of the ventricle. These granule-containing cells were immunohistochemically stained by applying the avidin-biotin-peroxidase complex method using an antiserum against -human atrial natriuretic polypeptide (ANP). Immunoelectron microscopy of sections stained using the immunogold method indicated that these specific granules are storage sites of ANP. Furthermore, an impulse-conducting system consisting of immunoreactive cells was clearly distinguishable from nonimmunoreactive ventricular cardiocytes. We conclude that specific-granule-containing cells, i.e., ANP-producing cells, are located in both the atrial walls and the ventricular impulse-conducting system. The presence of ANP may be correlated with impulse conduction.     

Cryofixation processing is an excellent method to improve the retention of adrenomedullin antigenicity

We reappraised the precise immunohistochemical localization of adrenomedullin (AM) by means of the combined use of the catalyzed signal amplification (CSA) system and plunge freezing (PF)/freeze substitution (FS) for light microscopy or high-pressure freezing (HPF)/FS for electron microscopy, focusing on the rat adrenal gland and heart. In the case of adrenal glands, the PF processing showed that almost all medullary cells were intensively immunoreactive, while the cortical cells showed weak immunoreaction. In the heart, almost all cardiac muscle cells of the atria were also vividly stained with the PF/FS and the CSA enhancement. On the contrary, traces of immunoreactions were seen in most of the ventricular cells. These results are consistent with the previous reports of AM radioimmunoassays and the expression of AM mRNA. However, the chemical fixation processing revealed heterogeneous immunostaining in the atrial and ventricular myocardium as well as the adrenal medulla. Intensity of the immunostaining in the chemically fixed tissues was not likely to correspond with that of AM radioimmunoassays. The HPF/FS processing clearly demonstrated the immunogold labeling on secretory granules of adrenal medullary cells as well as cardiac muscle cells of the right auricles. Immunogold labeling intensity of the cryofixed specimens was 3- to 25-fold higher than that of the chemically fixed ones.     

Localization and coexistence of atrial natriuretic peptide (ANP) and neuropeptide Y (NPY) in vertebrate adrenal chromaffin cells immunoreactive to TH,DBH and PNMT

Antisera specific for mammalian atrial natriuretic peptied (ANP) and neuropeptide Y (NPY) were applied to examine, in immunofluorescence, the occurrence of cells immunoreactive to ANP and NPY in the adrenal organs of mammals, birds, reptiles, amphibians, and bony fish. Catecholamine-containing cells were identified using antisera against tyrosine-hydroxylase, dopamine--hydroxylase, and phenylethanolamine-N-methyl-transferase. In all vertebrates studied, immunoreactivities to ANP and NPY occurred in adrenal chromaffin cells but were absent from the cortex or its homolog, the interrenal. The majority of immunoreactivities to ANP and NPY was confined to the adrenaline cells. In mammals, the number of ANP-immuno-reactive cells (60%–80% of the total cell population) exceeded that of the NPY-immunoreactive cells (35%–45%). In birds, reptiles, and Amphibia, the numbers of ANP-immunoreactive (35%–40%) and NPY-immunoreactive (30%–35%) cells were in a similar range. The bony fish showed a density of both ANP-immunoreactive (80%–90%) and NPY-immunoreactive (35%–40%) cells. In all species studied, immunoreactivities to ANP and NPY partially coexisted. Generally, 30%–55% of the ANP-immunoreactive cells also contained NPY-immunoreactivity. In rat, coexistence amounted to almost 100% and in quail to 95%. Except for the rat, three subpopulations of chromaffin cells seemed to occur: ANP-immunoreactive non-NPY-immunoreactive, ANP-immunoreactive+NPY-immunoreactive and NPY-immunoreactive non-ANP-immunoreactive cells. Thus, adrenal ANP and NPY share a conservative history and coexist as early as at the level of bony fish. The endocrine actions of ANP and NPY derived from medullary cells on cortical cells as found in mammals might be based on an ancestoral paracrine system. In submammalians, ANP and NPY may not only act as endocrine hormones, but also influence steroid-producing interrenal cells in a paracrine manner, and act as modulators on chromaffin cells.Dedicated to Professor dr. Angela Nolte (Münster, Germany) on the occasion of the 50th anniversary of her Ph.D. graduation     

Atrial natriuretic peptide detected by immunocytochemistry in peripheral organs of Tupaia belangeri

ANP (atrial natriuretic peptide), a peptide found in granules of mammalian atrial cardiac myocytes, has been shown to be active in regulation of blood pressure and body water homeostasis. The existence of ANP in atrium, pituitary, adrenal gland, and kidney of the rat had been immunocytochemically demonstrated with an antibody against rat ANP (102-126). We used the same antibody in immunocytochemical studies for the detection of ANP in peripheral organs of the tree shrew (Tupaia belangeri). The antibody stained granules in myocytes of cardiac atria which indicated that it reacted with tree shrew ANP. In contrast to the rat, no immunoreactive cells were found in pituitaries and adrenal glands. However, in the kidneys distal tubules in outer medulla and cortex were labeled. Ascending limbs of distal tubules were intensely stained when either the peroxidase-antiperoxidase (PAP) or the indirect immunofluorescence method were used. Collecting ducts and convoluted distal tubules in the outer cortex showed a granular type of staining when the immunofluorescence method was used. These data indicate that ANP is present in epithelial cells of distal tubules and collecting ducts, where it may be involved in the regulation of renal salt excretion.     

Atrial natriuretic peptide detected by immunocytochemistry in peripheral organs of Tupaia belangeri

Summary ANP (atrial natriuretic peptide), a peptide found in granules of mammalian atrial cardiac myocytes, has been shown to be active in regulation of blood pressure and body water homeostasis. The existence of ANP in atrium, pituitary, adrenal gland, and kidney of the rat had been immunocytochemically demonstrated with an antibody against rat ANP (102–126). We used the same antibody in immunocytochemical studies for the detection of ANP in peripheral organs of the tree shrew (Tupaia belangeri). The antibody stained granules in myocytes of cardiac atria which indicated that it reacted with tree shrew ANP. In contrast to the rat, no immunoreactive cells were found in pituitaries and adrenal glands. However, in the kidneys distal tubules in outer medulla and cortex were labeled Ascending limbs of distal tubules were intensely stained when either the peroxidase-antiperoxidase (PAP) or the indirect immunofluorescence method were used. Collecting ducts and convoluted distal tubules in the outer cortex showed a granular type of staining when the immunofluorescence method was used. These data indicate that ANP is present in epithelial cells of distal tubules and collecting ducts, where it may be involved in the regulation of renal salt excretion.     

Neuropeptide Y-like immunoreactivity in adrenaline cells of adrenal medulla and in tumors and plasma of pheochromocytoma patients

The occurrence of neuropeptide Y (NPY)-like immunoreactivity (LI) in the adrenal gland of several species as well as in tumor tissue and plasma from pheochromocytoma patients was investigated. NPY-LI was present in chromaffin cells of the adrenaline type in all species investigated except in the pig, as demonstrated by a colocalization of NPY-LI and the adrenaline-synthetizing enzyme phenylethanolamine N-methyltransferase (PNMT). NPY-LI in the adrenaline cells of the cat was clearly separated from the neurotensin-LI in the noradrenaline dopamine-beta-hydroxylase-positive, PNMT-negative cells. NPY-LI seems to co-exist with enkephalin-like material in the chromaffin cells. In addition, NPY-LI was present in nerves both within the adrenal cortex and medulla. The highest levels of NPY-LI were found in mouse and cat, while only a very low amount of NPY-LI was present in the pig adrenal. Characterization of the adrenal NPY-LI by reversed-phase high-performance liquid chromatography revealed that the main peak was similar to porcine NPY. In addition, two minor peaks of NPY-LI were present. High levels of NPY-LI were found in plasma and tumors from the pheochromocytoma patients. During manipulation of the tumors upon surgical removal, there was a marked increase in plasma NPY-LI in parallel with the raise in catecholamines and in blood pressure. At least two forms of NPY-LI were present in plasma and tumor extracts from pheochromocytoma patients with the main peak corresponding to porcine NPY. Since NPY exerts vasoconstrictor effects, it may be postulated that NPY contributes to the adrenal cardiovascular response and to the hypertension seen in pheochromocytoma patients.