Immunocytochemical localization of cAMP and cGMP in cells of the rat carotid body following natural and pharmacological stimulation

Summary Although the chemoreceptive function of the carotid body has been known for many decades, the cellular mechanisms of sensory transduction in this organ remain obscure. Common elements in the transductive processes of many cells are the cyclic nucleotide second messengers, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Studies from our laboratory have revealed stimulus-induced changes in cyclic nucleotide levels in the carotid body as measured by RIA, but such changes in second messenger levels have not been localized to specific cellular elements in the organ. The present immunocytochemical study utilized the avidin-biotin-peroxidase method to investigate the distribution of cAMP and cGMP in the rat carotid body and to assess changes in the intensity of immunostaining following in vitro stimulation by hypoxia, forskolin, sodium nitroprusside, high potassium, and atrial natriuretic peptide. Both cAMP and cGMP immunoreactivity were localized to type I cells of organs maintained in vivo and fixed by perfusion. Organs exposed to 100% O₂-equilibrated media in vitro produced low but visible levels of cAMP immunoreactivity in a majority of type I cells; hypoxia (5% O₂-equilibrated media) for 10 min moderately increased the level of immunoreactivity; forskolin (10^–5 M), or forskolin combined with hypoxia, dramatically increased cAMP levels in virtually all cells. Moderate levels of cGMP immunoreactivity in control carotid bodies in vitro were strikingly reduced by hypoxia; a significant increase in cGMP levels occurred following incubation in high potassium (100 mM), and under these conditions, the decrease in cGMP immunoreactivity with hypoxia was much more pronounced. The synthetic analog of atrial natriuretic peptide, atriopeptin III (10^–7 M), greatly elevated cGMP immunoreactivity in the type I cells. On the other hand, sodium nitroprusside (1 mM) elevated cGMP staining mostly in vascular elements of the carotid body in vitro. The data implicate the involvement of cyclic nucleotides in transduction of natural chemosensory stimuli by the type I cells in rat carotid body.     

Immunofluorescent localization of cGMP,cGMP-dependent protein kinase,calmodulin and cAMP in the rat uterus

Cyclic guanosine 3',5' monophosphate (cGMP), cGMP-dependent protein kinase, calmodulin and cyclic adenosine 3',5' monophosphate (cAMP) were localized in the uterus of the immature rat by an indirect immunofluorescence technique. cGMP, cGMP-dependent protein kinase and calmodulin were detected predominantly along epithelial and myometrial plasma membranes and in the adjacent cytoplasm. In contrast, cAMP immunoreactive material was found principally in the cytoplasm of connective tissue. After administration of 17 beta-estradiol, similar time-dependent changes were observed in the localization of cGMP, cGMP-dependent protein kinase and calmodulin in all uterine cell types. For the three compounds, nucleolar-like distribution of the immunofluorescence appeared approximately 12 h after treatment. A more dispersed, reticular distribution of the nuclear fluorescent staining was observed 20-24 h after hormonal treatment. Estrogen did not affect the localization of cAMP. The simultaneous mobilization of cGMP, cGMP-dependent protein kinase and calmodulin towards the same nuclear loci suggests concerted roles for these three molecules in nuclear metabolic processes during the development of the uterotrophic action of estrogens.     

Immunocytochemical localization of the receptor for asialoglycoprotein in rat liver cells

We used high-resolution immunocytochemistry on ultrathin frozen sections labeled with colloidal gold to study the subcellular distribution of the asialoglycoprotein receptor in rat liver. The receptor was localized along the entire hepatocyte plasma membrane, including the bile capillary membrane, but was scarce intracellularly. Sinusoidal lining (Kupffer) cells and blood cells showed no immunoreactivity. In liver cells of rats injected with 1 to 100 micrograms of asialoorosomucoid (ASOR) 2-15 min before tissue fixation, endocytotic internalization of receptors at the blood front was conspicuous. At all times in this interval, receptor was present in approximately 100-nm vesicles and larger vacuoles adjacent to the sinusoidal plasma membrane. No other significant intracellular receptor was noted during the 15-min exposure to ASOR; in particular, lysosomes and Golgi complex were not labeled. Our observations, in combination with data from the literature which demonstrate that, under these conditions, the ligand is transferred further to the Golgi complex-lysosome region, suggest that the receptor and ligand are dissociated in the vicinity of the plasma membrane, after which the receptor rapidly returns to the cell surface.     

The increase in cAMP and cGMP levels in the oestrogenized rat hypophysis.

Male and female rats were given oestradiol benzoate (1 mg s.c. twice a week for 3 weeks) and/or sodium nitroprusside (SN), a donor of nitric oxide (NO), which was administered in their food in amounts of 0.2 or 0.6 mg/rat/day. Neither oestradiol-induced hypertrophy of the hypophysis, nor the serum prolactin (PRL) level, was affected by the simultaneous administration of SN. The PRL content of the hypophysis rose after oestradiol in the males, but the increase was again uninfluenced by the simultaneous administration of SN and the cAMP content of the hypophysis--raised after oestradiol--was likewise unaffected. The amount of cGMP in the hypophysis after oestradiol rose only in males. Both the serum and the hypophyseal prolactin level were found to be correlated to the cAMP and the cGMP content of the hypophysis. It was found that the simultaneous administration of SN together with oestradiol slightly reduced the increase in the cGMP content of the hypophysis elicited with oestradiol treatment only.     

Immunocytochemical localization of cathepsin D in the rat osteoclast.

We performed immunocytochemical localization of cathepsin D in osteoclasts of the proximal growth plate of the rat femurs using both the avidin-biotin-peroxidase complex method for cryo-semi-thin (1 micron) sections and the colloidal gold-labeled IgG method for K4M ultra-thin sections. At the light microscopic level, cathepsin D immunoreactivity in the osteoclasts appeared at the vesicles, granules, and/or small vacuoles. They were distributed throughout the cytoplasm of each cell and were relatively numerous close to the bone surface. This antigen could not be detected at the eroded bone surface. As for other cells, immunoreactivity was seen only in the lysosomes of osteoblast-like cells. Immunoreactivity in the osteoclasts was stronger and greater in the density and number than in osteoblast-like cells. At the electron microscopic level, osteoclasts with well-developed ruffled border possessed numerous cathepsin D-containing lysosomes, vacuoles, and coated vesicle-like structures. Cathepsin D-containing lysosomes fused with cathepsin-negative vacuoles and formed large secondary lysosomes. Osteoclasts with poorly developed ruffled border possessed fewer cathepsin D-containing lysosomes than those with well-developed ruffled border. No immunogold particles were seen in vacuole-like channel expansions of the ruffled borders, between the channels of the ruffled borders, or on the eroded bone surface. These findings demonstrate that osteoclasts contain a large amount of cathepsin D. They suggest that cathepsin D is necessary for osteoclastic bone resorption, that it plays an indirect rather than direct role.     

Immunocytochemical study on the localization of neuron-specific enolase and S-100 protein in the carotid body of rats

Light- and electron-immunocytochemical investigation with the peroxidase-antiperoxidase (PAP) procedure revealed neuron-specific enolase and S-100 protein-like immunoreactivities specifically localized in the chief cells and the sustentacular cells of the rat carotid body, respectively. This finding suggests a neuron-like nature of the chief cells and a glia-like nature of the sustentacular cells on both embryological and functional bases.     

Changes in cGMP and cAMP content in the estrogen-stimulated rat uterus: temporal relationship with other parameters of hormonal stimulation.

The effect of estradiol-17beta, estriol, diethylstilbestrol and nafoxidine on rat uterine cGMP content was studied in relation with their respective estrogenic potency. Confirming previous results from Kuehl et al. (5), we observed a rise in uterine cGMP and a simultaneous decrease in cAMP content in treated animals. The reverse effect was obtained in the vagina after stimulation with estradiol or estriol. In the uterus, all compounds tested induced two main waves of cGMP increase corresponding to the two main phases of the estrogenic response i.e. the early fluid imbibition and the later period of true growth. No direct relationship could be established between the late rise in cGMP and true growth responses. A causal link between the first accumulation of uterine cGMP and the wet weight response in the early phase of estrogenic action is suggested by the comparison of time-course effects of the different compounds used on those two parameters.     

Immunocytochemical localization of ACTH-like immunoreactivity in rat adrenal glomerulosa and fasciculata cells

The role of ACTH in the synthesis of the adrenocortical hormones has been largely described. In order to investigate the localization of this peptide at the subcellular level of the adrenal glomerulosa and fasciculata cells, an immunocytological method was used. Rat adrenals were fixed and frozen. Ultrathin sections obtained by cryoultramicrotomy, were incubated with anti-beta (1-24) ACTH or anti-alpha (17-39) ACTH sera. The antigen-antibody reaction was detected by PAP complexes (revealed by 4-chloro-1-naphthol) or with protein A-colloidal gold or IgG-colloidal gold. The results obtained were the same whatever the antisera of the technique employed. All the cells of the adrenal zona glomerulosa and zona fasciculata were labelled. ACTH-like immunoreactivity in zona glomerulosa and zona fasciculata cells was observed at the plasma membrane level, in cytoplasmic matrix, mitochondria and nucleus (in the euchromatin close to the heterochromatin aggregations and, occasionally, associated with the nucleolus). No immunoreactivity was observed when non-immune serum or anti-ACTH serum preincubated with ACTH were used, nor there was any modification of the immunocytochemical reaction when anti-ACTH serum incubated with heterologous antigens was employed. These data, demonstrate the presence of endogenous ACTH in both adrenal glomerulosa and fasciculata cells, and suggest that the peptide is internalized after binding to the plasma membrane.     

Immunocytochemical localization of ACTH-like immunoreactivity in rat adrenal glomerulosa and fasciculata cells

Summary The role of ACTH in the synthesis of the adrenocortical hormones has been largely described. In order to investigate the localization of this peptide at the subcellular level of the adrenal glomerulosa and fasciculata cells, an immunocytological method was used. Rat adrenals were fixed and frozen. Ultrathin sections obtained by cryoultramicrotomy, were incubated with anti-(1–24) ACTH or anti (17–39) ACTH sera. The antigen-antibody reaction was detected by PAP complexes (revealed by 4-chloro-1-naphthol) or with protein A-colloidal gold or IgG-colloidal gold.The results obtained were the same whatever the antisera of the technique employed. All the cells of the adrenal zona glomerulosa and zona fasciculata were labelled. ACTH-like immunoreactivity in zona glomerulosa and zona fasciculata cells was observed at the plasma membrane level, in cytoplasmic matrix, mitochondria and nucleus (in the euchromatin close to the heterochromatin aggregations and, occasionally, associated with the nucleolus). No immunoreactivity was observed when non-immune serum or anti-ACTH serum preincubated with ACTH were used, nor there was any modification of the immunocytochemical reaction when anti-ACTH serum incubated with heterologous antigens was employed. These data, demonstrate the presence of endogenous ACTH in both adrenal glomerulosa and fasciculata cells, and suggest that the peptide is internalized after binding to the plasma membrane.T. Garcia-Caballero has a Postdoctoral Fellowship from Xunta de Galicia.     

The renin-angiotensin system in the rat brain. Immunocytochemical localization of angiotensinogen in glial cells and neurons

The distribution of angiotensinogen containing cells was determined in the brain of rats using immunocytochemistry. Specific angiotensinogen immunoreactivity is demonstrated both in glial cells and neurons throughout the brain, except the neocortical and cerebellar territories. Positive neurons are easily and invariably detected in female brains, and haphazardly in male brain (sex hormone dependent). Angiotensinogen immunoreactivity in male brain neurons can be induced by water deprivation or binephrectomy in some areas and particularly in paraventricular nuclei. Finally, the highest concentrations of positive neurons are found in the anterior and lateral hypothalamus, preoptic area, amygdala and some well known nuclei of the mesencephalon and the brainstem. Our results confirm the wide distribution of angiotensinogen mRNA in the brain reported recently by Lynch et al. (1987). Thus the demonstration of angiotensinogen in neurons and glial cells allows a greater understanding of the biochemical and physiological data in accordance with multiple brain renin angiotensin systems.     

雌激素Beta受体在大鼠脑内表达的免疫组化定位研究

为了探讨雌激素作用于神经系统的机理,采用硫酸镍铵增强显色的免疫组化SP法研究了新的雌激素受体（ER－β）在成年雌雄大鼠脑内的分布。研究证实ER－β免疫阳性物质主要位于神经元的细胞核内,但在个别脑区也可在胞浆甚至突起内检测到。最强的ER－β免疫阳性信号见于前嗅核、大脑皮质、小脑浦肯野细胞、斜角带垂直部、蓝斑和三叉神经运动核等部位;中等强度的染色见于隔内侧核、杏仁外侧核、黑质、中央灰质等部位;较弱的阳性反应见于下丘脑与杏仁复合体的部分核团。在一些部位还存在表达水平甚至细胞内定位模式的性别差异,如前庭上核内的表达只见于雌性;雄性大鼠三叉神经运动核内ER－β蛋白主要表达于胞浆内,细胞核为阴性;而在雌性大鼠该部位ER－β蛋白主要位于细胞核等。以上结果表明ER－β蛋白在大鼠脑内分布广泛并具有一定的性别差异,在与学习记忆有关的脑区如大脑皮质和基底前脑内有很高的表达,提示在脑组织内雌激素可能通过ER－β这一新的信号途径发挥多种重要的调控作用,如学习记忆等。     

Immunocytochemical identification and localization of immunoglobulin A within Paneth cells of the rat small intestine.

Light microscopic immunocytochemistry was used to identify Paneth cells by their lysozyme content and to detect immunoglobulin antigens within a subpopulation of these cells. Antisera specific for the heavy chains of rat or human immunoglobulin A and for immunoglobulin light chain antigens produced specific staining of rat Paneth cells. The distribution of immunoglobulin staining varied between adjacent Paneth cells in the same crypt and between Paneth cells in adjacent crypts, as well as between Paneth cell populations of different animals. No staining of rat Paneth cells was detected using antisera specific for the heavy chain of immunoglobulins G or M. The specific staining of Paneth cells for immunoglobulin A and light chain antigens was blocked by absorption of each antiserum with its respective purified antigen. Absorption of these antisera with purified rat lysozyme did not affect staining and thereby eliminated the possibility of immunologic cross-reactivity between lysozyme and immunoglobulin antigens. It is suggested, in light of current concepts of Paneth cell function, that the immunoglobulin staining of Paneth cells may reflect their ability to phagocytize immunoglobulin A-coated microorganisms or immune complexes containing immunoglobulin A.     

Immunocytochemical evidence for the specific localization of aldose reductase in rat Sertoli cells

Evidence that the enzyme aldose reductase (AR) is specifically located in Sertoli cells is presented by means of an established immunocytochemical technique and with a variety of approaches. By staining tissue sections, the enzyme was shown to be present in Sertoli cells at birth and the intensity of the immunocytochemical stain increased by 5 days of age to that found in the testes of older rats. By means of enzyme dispersion of mature testes; the culture of enriched Sertoli cell preparations from the testes of immature rats; and the collection of newly released testicular spermatozoa in rete testis fluid, it was shown that immunoreactive AR was not present in any testicular cell type except the Sertoli cell. The significance of the specific localization in Sertoli cells of a principal enzyme concerned in the sorbitol or polyol pathway for the conversion of aldose sugars to their corresponding ketoses is discussed.     

Immunocytochemical localization of aromatase in rat testis

Summary The immunocytochemical localization of armatase in the testes of young and adult rats was investigated by an indirect-immunofluorescent method using antihuman placental aromatase-II cytochrome P-450 antibody. In both young (1 and 2 weeks old) and adult rats, only the Leydig cells in the interstitial tissue showed a positive immunoreaction for aromatase, while the germ cells and Sertoli cells in the seminiferous tubule were entirely negative. In addition, electron microscopy revealed that the Leydig cells in the testes of young as well as adult rats have a well-developed smooth endoplasmic reticulum, mitochondria with tubulovesicular cristae, and a few lipid droplets; these structures being characteristic of steroid secretory cells. On the basis of these results, we suggest that estrogens are mainly synthesized in Leydig cells of the testes.Supported by grants from the Ministry of Education, Science, and Culture, Japan, and USPHS HD 04945     

Immunocytochemical localization of aromatase in rat testis

The immunocytochemical localization of aromatase in the testes of young and adult rats was investigated by an indirect-immunofluorescent method using antihuman placental aromatase-II cytochrome P-450 antibody. In both young (1 and 2 weeks old) and adult rats, only the Leydig cells in the interstitial tissue showed a positive immunoreaction for aromatase, while the germ cells and Sertoli cells in the seminiferous tubule were entirely negative. In addition, electron microscopy revealed that the Leydig cells in the testes of young as well as adult rats have a well-developed smooth endoplasmic reticulum, mitochondria with tubulovesicular cristae, and a few lipid droplets, these structures being characteristic of steroid secretory cells. On the basis of these results, we suggest that estrogens are mainly synthesized in Leydig cells of the testes.     

Immunocytochemical localization of pepsinogen in rat stomach

The localization of pepsinogen in rat stomachs was investigated by a postembedding immunoferritin method. When the preparations embedded in Epon were used, the secretory granules of chief cells were stained heavily and the granules of mucous neck cells were stained moderately. The secretory granules of cells intermediate between mucous neck cells and chief cells showed a bizonal staining; the electron dense parts were stained heavily and the electron lucent parts were stained moderately. The secretory granules of pyloric gland cells, on the other hand, were labeled faintly. However, the secretory granules of surface mucous cells, foveolar mucous cells, endocrine cells, cardiac mucous cells and cardiac serous cells were not stained by the method. The protein A-gold method showed a similar staining pattern of pepsinogen to that of the immunoferritin method. When the samples embedded in Lowicryl K4M were used to enhance the stainability of pepsinogen, essentially the same staining pattern as that of the samples embedded in Epon was obtained. In addition, the Golgi apparatus and the rough surfaced endoplasmic reticulum were more easily stained.     

Immunocytochemical localization of Shc and activated EGF receptor in early endosomes after EGF stimulation of HeLa cells.

After binding of epidermal growth factor (EGF), the EGF receptor (EGFR) becomes autophosphorylated via tyrosine. The ligand-activated receptor is internalized by endocytosis and subsequently degraded in the lysosomal pathway. To follow EGFR activation after EGF stimulation, we generated antisera to the EGFR phosphotyrosine sites pY992 and pY1173. The SH2 region of Shc binds to both these sites. Both antisera identified EGFR after EGF binding and did not crossreact with the unactivated receptor. The intracellular distribution of phosphorylated EGFR after ligand binding was traced by two-color immunofluorescence confocal microscopy and immunoelectron microscopy. Before EGF stimulation EGFR was primarily located along the cell surface. When internalization of activated EGFR was inhibited by incubation with EGF on ice, Y992- and Y1173-phosphorylated EGFR were located along the plasma membrane. Ten minutes after internalization at 37C, Y992- and Y1173-phosphorylated EGFR were almost exclusively located in early endosomes, as shown by co-localization with EEA1. Immunoelectron microscopy confirmed that phosphorylated EGFR was located in intracellular vesicles resembling early endosomes. After EGF stimulation, the adaptor protein Shc redistributed to EGFR-containing early endosomes. Our results indicate that EGFR activation of Shc via tyrosine-phosphorylated Y992 and Y1173 occurred in early endocytic compartments, and support a role for membrane trafficking in intracellular signaling.     

Immunohistochemical localization of tryptophan hydroxylase and serotonin transporter in the carotid body of the rat

It has been proposed that serotonin (5-HT) facilitates the chemosensory activity of the carotid body (CB). In the present study, we investigated mRNA expression and immunohistochemical localization of the 5-HT synthetic enzyme isoforms, tryptophan hydroxylase 1 (TPH1) and TPH2, and the 5-HT plasma membrane transport protein, 5-HT transporter (SERT), in the CB of the rat. RT-PCR analysis detected the expression of mRNA for TPH1 and SERT in extracts of the CB. Using immunohistochemistry, 5-HT immunoreactivity was observed in a few glomus cells. TPH1 and SERT immunoreactivities were observed in almost all glomus cells. SERT immunoreactivity was seen on nerve fibers with TPH1 immunoreactivity. SERT immunoreactivity was also observed in varicose nerve fibers immunoreactive for dopamine beta-hydroxylase, but not in nerve fibers immunoreactive for vesicular acetylcholine transporters or nerve terminals immunoreactive for P2X₃ purinoreceptors. These results suggest that 5-HT is synthesized and released from glomus cells and sympathetic nerve fibers in the CB of the rat, and that the chemosensory activity of the CB is regulated by 5-HT from glomus cells and sympathetic nerve fibers.