Lysosomal enzyme activities in hypo- and hypersecretory anterior pituitary cells

Summary The involvement of lysosomes in ACTH and prolactin secretion was studied. Lysosomes were visualized in the anterior pituitary by their non-specific esterase (gold thioacetic acid technique) or acid phosphatase (Gomori technique) activity. Corticotrophs and mammotrophs were identified by postembedding immunocytochemistry for their respective hormones. Corticotrophs were rendered hypersecretory by bilateral adrenalectomy (7 or 12 days prior to examination), hyposecretory by dexamethasone administration. Prolactin secretion was enhanced by 17-beta-estradiol, prolactin release was inhibited by bromoergocriptine administration. Long-term hypersecretion of ACTH was accompanied by the presence of numerous autophagic vacuoles often containing secretory granules in the corticotrophs. Lysosomal enzyme-containing tubules and small lysosomes were abundant in the cytoplasm near the cell membrane, among the mature secretory granules. Feed-back inhibition of ACTH release by dexamethasone resulted in the extension of enzyme-containing tubules, continuous with cisternae and small lysosomes anywhere in the cytoplasm and in the appearance of numerous crinophagic vacuoles. A higher frequency of tubular lysosomes was described at the periphery of mammotrophs stimulated by 17-beta-estradiol. Bromoergocriptine caused a high incidence of characteristic crinophagic vacuoles in the prolactin cells. The concept of crinophagy has been extended to the corticotrophs. Morphological phenomena were attributed to the traffic and increased turnover of membranes, ligands and cytoplasmic organelles during stimulated secretion.     

Lysosomes in anterior pituitary corticotrophs of the rat. A combined immunocytochemical and enzyme cytochemical study

E600 resistant non-specific esterase activity or acid phosphatase activity were localized in corticotrophic cells identified by postembedding immunocytochemistry (PAP of protein A-immunogold techniques). The lysosomal system of this cell type consists of dense bodies, of a population of small lysosomes mostly situated at the cell periphery in the vicinity of secretory granules as well as of tubular structures. These latter were located either in the central part of the cytoplasm and probably belonged to the Golgi apparatus or at the cell periphery, partly in the extensions. Small lysosomes occurred to be in continuity with enzyme-containing tubules. In a few structures lysosomal enzyme activity and ACTH immunoreactivity overlapped. Some autophagic vacuoles seemed to contain secretory granule matrix. It is suggested that the concept of crinophagy can be extended to the corticotrophs, though the lysosomal system may be involved in the specific function of this cell type by other mechanisms as well.     

Morphometric analysis of the autophagic and crinophagic lysosomal systems in mammotropes throughout the estrous cycle of the rat

Summary The crinophagic and autophagic lysosomal systems were studied in mammotropes (prolactin secreting cells) of the adenohypophysis throughout the estrous cycle of the rat. By means of morphometric analysis, it was found that the volume of secondary autophagic lysosomes was usually greater than that of the crinophagic type. Although the volumes of both secondary autophagic and crinophagic lysosomes were minimal throughout proestrus and diestrus 2, the autophagic lysosomal volume per mammotrope was elevated during the estrous period. The volume of secondary crinophagic lysosomes per mammotrope increased during late estrus and remained elevated throughout early diestrus 1. Furthermore, there was an inverse relationship between the volume of mature secretory granules per cell and of the crinophagic system. These data suggest a role for lysosomes in the regulation of synthesis and secretion of prolactin by the adenohypophysis of the rat.Supported by grant HD 11571 from the National Institutes of Health     

溶酶体在垂体—肾上腺皮质激素分泌调节中的作用

本实验用地塞米松造成大鼠垂体促皮质激素细胞及其靶腺肾上腺皮质束状带细胞分泌抑制,对这两种细胞中的溶酶体及分泌自噬和自体吞噬活动进行了超微结构观察、CMP 酶细胞化学定性和形态计量。实验结果显示,在分泌受抑制状态下,垂体促皮质激素细胞中分泌自噬和自体吞噬作用加强,与此同时,肾上腺皮质细胞中自体吞噬作用也业著加强。这些结果表明,在分泌类固醇激素的细胞中,溶酶体以自体吞噬的方式清除一部分生产激素的细胞器,可能是一种普遍存在的分泌调节机制,正如在分泌蛋白质和肽类激素的细胞中普遍存在着分泌自噬这一调节机制一样。     

Corticotroph cells in primary cultures of rat adenohypophysis: A light and electron microscopic immunocytochemical study

Summary Monolayer cultures of trypsin-dispersed cells of the rat adenohypophysis were grown for 5 to 54 days. ACTH was localized by immunocytochemistry using an antiserum to synthetic ACTH^1–28 prepared in rabbit and sheep anti-goat immunoglobulin coupled with peroxidase. ACTH content of the culture medium was measured by radioimmunoassay.Corticotrophs were found in the cultures and ACTH in the medium at each cultivation time. The corticotrophs retained their essential morphological characteristics. Immunological staining was found in the secretory granules, some tubular or saccular structures, parts of the rough endoplasmic reticulum, and the cytoplasmic matrix. Immature secretory granules in the Golgi apparatus as well as some Golgi elements showed different degrees of immunoreactivity. In agreement with the high ACTH content of the culture medium the number, size and shape of the secretory granules, the active Golgi apparatus, the high amount of extragranular ACTH as well as pictures suggesting granule extrusion claim for a high ACTH synthesis and transport (and low ACTH storage) in the cultured corticotrophs.     

Glycoconjugate localization with lectin and PA-TCH-SP cytochemistry in rat hypophysis

Glycoconjugates were localized by light microscopy with lectin-peroxidase conjugates and by electron microscopy with the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) sequence in immunocytochemically or morphologically identified cell types in rat pituitary. Lectin histochemistry demonstrated sialic acid and glycoconjugates with N-glycosidically linked oligosaccharides in gonadotrophs, thyrotrophs, and corticotrophs. Galactose penultimate to sialic acid was observed mostly in gonadotrophs. The terminal galactose-N-acetylgalactosamine disaccharide was detected in a few gonadotrophs and in a moderate number of mammotrophs. Fucose was localized in only corticotrophs with two fucose-binding lectins and in thyrotrophs with another. Several different monosaccharides were seen in glycoconjugates in melanotrophs and in Herring bodies. Melanotrophs displayed heterogeneous staining with fucose-binding lectins. A small number of nonsecretory cells were also visualized in the pars distalis by virtue of their glycogen content. PA-TCH-SP staining revealed complex carbohydrates in secretory granules and some Golgi cisternae in all types of hormone-producing cells in the pars distalis except for the somatotrophs. Melanotrophs of pars intermedia exhibited stained secretory granules and irregular dense bodies containing a stained meshwork. Corticotrophs of the pars distalis lacked the latter bodies, although they form the same glycoprotein precursor hormone as melanotrophs. Lectin conjugates and the PA-TCH-SP sequence stained some groups of secretion granules in Herring bodies, possibly representing vasopressin-containing granules as well as other cell types in the pars nervosa.     

Immunocytochemical localization of cathepsin B in rat anterior pituitary endocrine cells, with special reference to its co-localization with renin and prorenin in gonadotrophs

We examined by immunocytochemistry the localization of cathepsin B in endocrine cells of rat anterior pituitary lobe, using a monospecific antibody to cathepsin B. By light microscopy, granular immunodeposits for cathepsin B were detected in most endocrine cells of anterior pituitary lobe. Cells immunoreactive for luteinizing hormone (LH) were diffusely immunostained by anti-cathepsin B. By electron microscopy, immunogold particles for cathepsin B were localized in lysosomes of thyrotrophs, somatotrophs, and mammotrophs. In mammotrophs, immunogold particles for cathepsin B were also detected in crinophagic bodies. Double immunostaining co-localized immunogold particles for LH and cathepsin B in secretory granules of gonadotrophs. Immunocytochemistry was also applied to demonstrate localization of renin and prorenin in LH-producing gonadotrophs; immunogold particles for renin were co-localized with those for LH, cathepsin B, or prorenin in their secretory granules. Immunogold particles for prorenin were also co-localized with those for LH or cathepsin B in secretory granules, but prorenin-positive granules appeared less frequently than renin-positive granules. These results suggest that cathepsin B not only plays a role in the protein degradation in lysosomes of anterior pituitary endocrine cells but also participates in the activation of renin in gonadotrophs, as has been demonstrated in secretory granules of juxtaglomerular cells.     

Some functional aspects of canine corticotrophs

To examine the regulation and functional significance of canine pituitary pars intermedia corticotrophs, ACTH and cortisol responses to CRF were studied in healthy dogs before and after treatment with dexamethasone. In addition the effects of the dopamine agonist bromocriptine and the dopamine antagonist pimozide were investigated. In the latter two instances prolactin concentrations were also measured. Finally the pituitaries were studied immunocytochemically for ACTH and alpha-MSH. No response of ACTH or cortisol to bromocriptine was observed. Pimozide caused a slight rise in ACTH levels in some dogs. However, prolactin levels significantly decreased with bromocriptine and increased with pimozide. Injection of synthetic ovine CRF to dogs was followed by sharp increases in ACTH and cortisol values. These responses were obliterated by prior treatment with dexamethasone. In 1 of 4 dogs given dexamethasone before euthanasia, there were few pars distalis cells with ACTH(1-24) immunopositivity, although persistence of ACTH(1-24) reaction was noted within cells of the pars intermedia. The results indicate that none of the CRF-induced ACTH secretion in dogs is derived from pars intermedia corticotrophs. Dosages of bromocriptine and pimozide that clearly alter prolactin secretion do not consistently affect ACTH levels.     

Corticotrophs and peptides

Corticotrophs were long thought to be a static, homogeneous population of cells that respond positively to hypothalamic stimulation, are inhibited by glucocorticoid feedback and secrete a single biologically active peptide, ACTH(1-39). Our current understanding is that this is an oversimplification and corticotrophs are a dynamic and more complex group of cells. The biosynthetic precursors of ACTH and other cleavage products of proopiomelanocortin (POMC) have been found to be secreted by anterior pituitary cells, to circulate and to have biological activity. POMC and the biosynthetic intermediate, pro-ACTH, exert activity antagonistic to ACTH(1-39) on glucocorticoid secretion by adrenal cells, and other derivatives of POMC are mitogenic to adrenocortical cells. In terms of responses to hypothalamic and peripheral factors, corticotrophs are functionally heterogeneous. This is reflected in the sensitivity of individual subtypes of corticotrophs to CRH, vasopressin and glucocorticoids. There is a functional plasticity amongst the various types of corticotrophs. During gestation, in fetal sheep, changes occur in the overall ACTH-secretory responses to CRH relative to vasopressin, the proportions of total corticotrophs that respond to the respective peptides and the average secretory response of individual cells. Corticotrophs also respond to locally produced pituitary factors. Local actions of leukaemia inhibitory factor are demonstrated by the effects of immunoneutralization of the peptide in pituitary cells. Urocortin and preproTRH(178-199) are locally produced peptides with potent stimulatory and inhibitory actions on corticotrophs, respectively. The specific roles of these peptides are under investigation.     

Human pituitary corticotrophs: their amphophilic stainability and immunohistochemical characterization

Immunohistochemical characterization of the human pituitary beta(R) cells was investigated through the findings of the immunoreactivities with anti-porcine ACTH, -rat TSH, -rat FSH sera. Immunostained corticotrophs are oval or round in shape and localized in the anteromedial wedge. It is shown on the adjacent sections that they correspond to the beta(R) cells with amphophilic stainability with PAS-iron hematoxylin. In this wedge, amphophilic cells are preponderant, but PAS-positive thyrotrophs and gonadotrophs are not numerous. Amphophilic stainability varies in degree from cell to cell: One cell contains numerous medium-size of secretory granules weakly stained with iron hematoxylin and strongly with PAS in the PAS-positive cytoplasm, and the other cell is filled with big secretory granules intensively stained with iron hematoxylin and weakly with PAS. The immunostained TSH, LH and FSH cells are different from the beta(R) corticotrophs, because anti-ACTH serum never reacts to the TSH, LH and FSH cells in the two adjacent sections. LH and FSH reactivities are observed in the single cells. It is concluded that human corticotrophs are amphophilic beta(R) cells filled with secretory granules, and that they have quite a different appearance from the rat chromophobic stellate corticotrophs with a row arrangement of secretory granules along the plasma membrane.     

Ultrastructure of the cell types of the anterior hypophysis in a lizard. I. Corticotrophs

Corticotrophs of the teiid lizard Cnemidophorus lemniscatus are situated in the rostral zone of the pars distalis. In normal animals, they are usually rounded cells with slightly eccentric vesicular nuclei, especially characterized by a lucent hyaloplasm and medium-sized secretory granules of uniform high density. Granules are almost spherical, with small angular deformations, and closely bounded by a fuzzy membrane. Many cells have only a few or a moderate number of granules, with large areas of cytoplasm devoid of them; in others, granules fill the supranuclear region. The cytoplasm exhibits numerous ribosomes, often in rosettes and mostly free, a series of loosely superimposed cisternae of rough endoplasmic reticulum, small dictyosomes, and elongate mitochondria of light matrix. Metyrapone administration during 2-8 days causes dramatic alterations in corticotrophs; they become hypertrophic and extensively degranulated, with a great development of the endoplasmic reticulum and Golgi apparatus, eventually showing a row of large peripheral granules of uneven structure, enclosed in ample vesicles studded with ribosomes. A lesser degree of hypertrophy and degranulation of corticotrophs appears during the first two weeks after thyroidectomy or gonadectomy, and may be partially attributed to surgical stress. Well granulated enlarged corticotrophs, with hypertrophic endoplasmic reticulum and Golgi apparatus, are probably a result of hormonal imbalance in lizards of both sexes gonadectomized for one or two months.     

Changes with senescence in the fine structure of the granular convoluted tubule of the submandibular gland of the mouse

Cells of the granular convoluted tubules (GCTs) of the submandibular gland of senescent male mice show structural changes indicative of functional decline. In order to define the nature of these age-related changes more clearly, the fine structure of GCT cells of 12- and 28-month-old males was compared. In old mice, there was cell-to-cell variation in the extent of these changes, with some cells of senescent males appearing no different from those of young adults. In affected cells the most striking alterations were seen in secretion granules and lysosomal elements. Secretion granules varied greatly in size, with some GCT cells having only very fine apical granules. Secondary lysosomes and large lipofuscin granules were frequent in the basal cytoplasm. Very large dense bodies (3-5 micron) occurred in many cells. These possibly represent intracellular pools of released secretory materials, as they were occasionally seen in continuity with the luminal contents. Structures whose appearance was intermediate between the very large dense bodies and lipofuscin granules were common, suggesting crinophagic activity. There was an apparent decrease in numbers of polysomes and in the extent of the Golgi apparatus. These fine structural changes are consistent with impairments with advanced age in synthesis and posttranslational processing of secretory products by affected GCT cells. In addition to cell-to-cell variation in any one male, there was also interanimal variation in the degree and extent of these senescent changes.     

Prolactin crinophagy is induced in the estrogen-stimulated male rat pituitary

The phenomenon of crinophagy in rat pituitary mammotrophs, or lysosomal uptake of prolactin secretory granules, was confirmed by means of double-label immunogold electron microscopy, and shown to be induced in estrogen-stimulated male rats. Rabbit antibodies to rat cathepsin D were used to label lysosomes, and to rat prolactin to label secretory granules. The pituitaries were fixed in 4% formaldehyde and 1% glutaraldehyde, embedded in Lowicryl K4M, and thin sections were exposed successively to primary antibodies, biotin-labelled second antibodies, and streptavidin-gold, with an amplification procedure for cathepsin D. Cathepsin D and prolactin were detected separately on opposite sides of the sections, using 5-nm and 15-nm gold particles. Lysosomal uptake of prolactin secretory granules was not observed in untreated control rats. It was detected in about 26% of lysosome-containing mammotroph cell sections in estrogen-stimulated rats and at 7 h after estrogen withdrawal, but fell to 14% at 24 h and to 2% at 72 h after estrogen withdrawal.     

Prolactin crinophagy is induced in the estrogen-stimulated male rat pituitary

Summary The phenomenon of crinophagy in rat pituitary mammotrophs, or lysosomal uptake of prolactin secretory granules, was confirmed by means of double-label immunogold electron microscopy, and shown to be induced in estrogen-stimulated male rats. Rabbit antibodies to rat cathepsin D were used to label lysosomes, and to rat prolactin to label secretory granules. The pituitaries were fixed in 4% formaldehyde and 1% glutaraldehyde, embedded in Lowicryl K4M, and thin sections were exposed successively to primary antibodies, biotin-labelled second antibodies, and streptavidin-gold, with an amplification procedure for cathepsin D. Cathepsin D and prolactin were detected separately on opposite sides of the sections, using 5-nm and 15-nm gold particles. Lysosomal uptake of prolactin secretory granules was not observed in untreated control rats. It was detected in about 26% of lysosome-containing mammotroph cell sections in estrogen-stimulated rats and at 7 h after estrogen withdrawal, but fell to 14% at 24 h and to 2% at 72 h after estrogen withdrawal.     

Cationic ferritin uptake by cultured anterior pituitary cells treated with the proteinase inhibitor, BOC-DPhe-Phe-Lys-H

Cultured cells from the anterior pituitary glands of adult rats were treated with the tripeptide aldehyde proteinase inhibitor, BOC-DPhe-Phe-Lys-H. The addition of this tripeptide aldehyde decreased the in vitro release of prolactin to 25% of the control value, while the release of growth hormone in the same cultures decreased to 33% of the control value. Prolactin immunostaining was stronger in semithin sections of proteinase-inhibitor-treated cultures than in control sections. After 2 h treatment with the inhibitor, prolactin- and growth hormone-containing secretory granules were numerous, and the number of crinophagic vacuoles had increased. In the presence of the inhibitor, the overall cytoarchitecture of parenchymal cells was well preserved, and the pathway of the uptake of cationic ferritin appeared to be unaffected.     

Intracellular sites of proteolytic processing of pro-opiomelanocortin in melanotrophs and corticotrophs in rat pituitary.

A synthetic peptide (ST-1) corresponding to the cleavage site between ACTH and beta-lipotropic hormone moieties of murine pro-opiomelanocortin (POMC) was constructed and its polyclonal antibody was generated. This antiserum immunoprecipitated only POMC from extracts of AtT-20 cells. Moreover, an antiserum raised against porcine ACTH immunoprecipitated both ACTH[1-39] and POMC. When ultra-thin frozen sections of melanotrophs in rat pars intermedia were immunolabeled with anti-ST-1 followed by protein A-gold, gold particles indicating the presence of POMC were selectively found in the electron-dense secretory granules in the Golgi area. In addition, the immunolabeling was also observed in the cisternae of the Golgi apparatus and rough endoplasmic reticulum. In contrast, with a polyclonal antibody specific for alpha-melanocyte-stimulating hormone the gold particles were found exclusively in the electron-lucent secretory granules, with none seen in the electron-dense secretory granules. With anti-ACTH serum, gold particles were observed in the electron-dense and -lucent secretory granules. In corticotrophs in the pars distalis, many gold particles indicating the presence of POMC were observed in the Golgi and peripheral secretory granules, but the percentage of immunolabeling in the peripheral secretory granules varied from cell to cell. On the other hand, ACTH immunolabeling was found in almost all the secretory granules. This finding suggests that the processing of POMC in corticotrophs might occur in the relatively peripheral granules. These results suggest that the intracellular sites of POMC processing are somewhat different between melanotrophs and corticotrophs in the pituitary.     

Plasminogen activators of the pituitary gland: enzyme characterization and hormonal modulation

We studied plasminogen activator (PA) of the rat pituitary gland in organ and cell monolayer culture. Both anterior and intermediate lobes contain, synthesize and secrete a mixture consisting of the two known types of PA: urokinase and so-called tissue PA. Both enzymes were formed essentially by all PA secreting cells, and PA was identified specifically in mammotrophs, corticotrophs, and luteinizing hormone containing gonadotrophs. Pituitary PA production was modulated on exposure to a variety of biological effectors: anterior lobe PA secretion was stimulated by agents that raised intracellular cAMP concentration; his process depended on de novo enzyme synthesis. Enzyme production was repressed by androgens and glucocorticoids. When anterior lobe cultures were maintained in plasminogen-free media, the extracellular, secreted forms of ACTH consisted almost exclusively of the high molecular weight forms (31,000 and 23,000); the smaller forms (13,000 and 4,500) were also found in the extracellular medium of cultures supplemented with plasminogen. In contrast, the size distribution of intracellular ACTH species was unaffected by the presence of plasminogen. These results resemble those previously obtained with pancreatic islets and are consistent with the possibility that plasmin, generated by PA secretion, participates in prohormone processing. PA synthesis in intermediate lobe explants was stimulated by exposure to dibutyryl cAMP, and repressed by hydrocortisone. In accordance with the dopaminergic control of intermediate lobe function in some vertebrates, apomorphine strongly repressed PA synthesis in intermediate, but not anterior lobe cultures.     

Sorting of three secretory proteins to distinct secretory granules in acidophilic cells of cow anterior pituitary

The distribution of three proteins discharged by regulated exocytosis--growth hormone (GH), prolactin (PRL), and secretogranin II (SgII)--was investigated by double immunolabeling of ultrathin frozen sections in the acidophilic cells of the bovine pituitary. In mammotrophs, heavy PRL labeling was observed over secretory granule matrices (including the immature matrices at the trans Golgi surface) and also over Golgi cisternae. In contrast, in somatotrophs heavy GH labeling was restricted to the granule matrices; vesicles and tubules at the trans Golgi region showed some and the Golgi cisternae only sparse labeling. All somatotrophs and mammotrophs were heavily positive for GH and PRL, respectively, and were found to contain small amounts of the other hormone as well, which, however, was almost completely absent from granules, and was more concentrated in the Golgi complex, admixed with the predominant hormone. Mixed somatomammotrophs (approximately 26% of the acidophilic cells) were heavily positive for both GH and PRL. Although admixed within Golgi cisternae, the two hormones were stored separately within distinct granule types. A third type of granule was found to contain SgII. Spillage of small amounts of each of the three secretory proteins into granules containing predominantly another protein was common, but true intermixing (i.e., coexistence within single granules of comparable amounts of two proteins) was very rare. It is concluded that in the regulated pathway of acidophilic pituitary, cell mechanisms exist that cause sorting of the three secretory proteins investigated. Such mechanisms operate beyond the Golgi cisternae, possibly at the sites where condensation of secretion products into granule matrices takes place.     

Cationic ferritin uptake by cultured anterior pituitary cells treated with the proteinase inhibitor,BOC-DPhe-Phe-Lys-H

Summary Cultured cells from the anterior pituitary glands of adult rats were treated with the tripeptide aldehyde proteinase inhibitor, BOC-DPhe-Phe-Lys-H. The addition of this tripeptide aldehyde decreased the in vitro release of prolactin to 25% of the control value, while the release of growth hormone in the same cultures decreased to 33% of the control value. Prolactin immunostaining was stronger in semithin sections of proteinase-inhibitor-treated cultures than in control sections. After 2 h treatment with the inhibitor, prolactin- and growth hormone-containing secretory granules were numerous, and the number of crinophagic vacuoles had increased. In the presence of the inhibitor, the overall cytoarchitecture of parenchymal cells was well preserved, and the pathway of the uptake of cationic ferritin appeared to be unaffected.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Use of immunoperoxidase and immunogold methods in studying prolactin secretion and application of immunogold labelling for pituitary hormones and neuropeptides

Two immunocytochemical methods, immunoperoxidase and immunogold (IG), were used in an attempt to study the dynamic process of prolactin release from stimulated rat pituitary mammotrophs. The immunogold method was also used to localize other pituitary hormones including growth hormone, follicle-stimulating hormone, luteinizing hormone, and the neuropeptides substance P, neuropeptide tyrosine, leu-enkephalin, and atrial natriuretic factor in peripheral nerves. Light-microscopic immunoperoxidase staining of prolactin revealed a unique distribution of immunoreactive mammotrophs. Two groups of cells were seen, one centrally located and one forming a narrow peripheral rim on the gland. The two groups were separated by a zone of nonimmunoreactive cells. In addition, the distribution of immunoperoxidase-stained material was not uniform in all mammotrophs. In some, prolactin immunoreactive material was clumped near the nucleus (in the Golgi cisternae); in others it was more diffused within the cytoplasm (but immediately surrounding the cisternae of rough endoplasmic reticulum). After stimulation of mammotrophs, via suckling, prolactin-immunoreactive material was visualized in extracellular spaces. With immunogold methods, prolactin labelling was seen mainly in secretory granules; but some labelling of Golgi cisternae and rough endoplasmic reticulum also occurred. Immunogold labelling revealed that material immunoreactive for leu-enkephalin and atrial natriuretic factor was present in nerve terminals in the rat paracervical ganglion. Material immunoreactive for substance P and neuropeptide tyrosine was present in nerve terminals in the guinea pig heart. Thus, in some situations the immunoperoxidase technique was useful and helped to visualize "grossly" the presence of specific antigens, but it was inadequate for fine ultrastructural localization of these antigens. The immunogold technique was excellent for precise localization of antigens and especially for the detection of colocalization of different antigens. This method can be used in very different structures, such as the adenohypophysis and peripheral nervous tissue, without any modification except for the nature of the antibodies.