Immunocytochemical analysis of chromaffin cell proliferation in vitro.

The bromodeoxyuridine (BrdU) incorporation technique for immunocytochemical labeling of S-phase nuclei was optimized for the study of chromaffin cell proliferation. Sequential fixation in ethanol followed by paraformaldehyde, and the use of DNAse to render incorporated BrdU accessible to antibody, permitted permanent double staining for BrdU and tyrosine hydroxylase. The efficacy of the technique was demonstrated in microcultures of dissociated neonatal rat adrenal glands, in which chromaffin cells exhibited proliferative responses to nerve growth factor and fibroblast growth factor similar to those previously demonstrated by autoradiography. Growth factor responsiveness was observed in both serum-containing and serum-free medium.     

Immunocytochemical localization of endothelin in cultured bovine endothelial cells

Summary To investigate the intracellular localization of endothelin in cultured endothelial cells, an immunocytochemical study was carried out by the post-embedding protein A-gold technique with endothelin-specific antiserum. Gold particles were seen on the rough endoplasmic reticulum, the Golgi cisternae, the Golgi vesicles, small vesicles beneath the cell membrane, and the lysosomes. By contrast, no secretory granules were observed. These results suggest that endothelin is secreted by a constitutive pathway and that the lysosome may play an important role in regulating the biological activity of endothelin.     

Immunocytochemical localization of endothelin in cultured bovine endothelial cells

To investigate the intracellular localization of endothelin in cultured endothelial cells, an immunocytochemical study was carried out by the post-embedding protein A-gold technique with endothelin-specific antiserum. Gold particles were seen on the rough endoplasmic reticulum, the Golgi cisternae, the Golgi vesicles, small vesicles beneath the cell membrane, and the lysosomes. By contrast, no secretory granules were observed. These results suggest that endothelin is secreted by a constitutive pathway and that the lysosome may play an important role in regulating the biological activity of endothelin.     

Retrieving vesicles in secretion-induced rat chromaffin cells contain fodrin

We examined the distribution of fodrin and cytochrome b561 in secretion-induced rat chromaffin cells (epinephrine cells) by immunofluorescence and immunoelectron microscopy. Fasted rats injected with a large dose of insulin were perfusion-fixed and frozen sections of the adrenal medulla were immunolabeled. Fodrin, a peripheral membrane protein, was distributed only in the cell periphery in control cells, but was observed in the cell interior after the insulin treatment; many of the markers were found around small vesicles, 50-200 nm in diameter, and large vacuoles, more than 500 nm in diameter. On the other hand, cytochrome b561, an integral membrane protein, was seen only in the chromaffin granules in control cells, and appeared in small vesicles in the stimulated cells but not in large vacuoles. By double immunolabeling it was shown that cytochrome b561 coexisted with fodrin in the small vesicles. The coexistence of the two proteins was confirmed by the labeling of subcellular particles immunoadsorbed from the insulin-treated adrenal medulla homogenate; vesicles immunoisolated with anti-fodrin antibody on polyacrylamide beads were positively immunolabeled with anti-cytochrome b561 antibody. The present results show that during massive secretion fodrin is taken into the cell interior by vesicles, which may be a mechanism that retrieves the secretory granule membrane from the cell surface.     

Immunocytochemical localization of secretory proteins in bovine pancreatic exocrine cells

The bovine exocrine pancreatic cell produces a variety of enzymes and proenzymes for export. Biochemical studies by Greene L.J., C.H. Hirs, and G.E. Palade (J. Biol. Chem. 1963. 238:2054) have shown that the mass proportions of several of these proteins in resting pancreatic juice and zymogen granule fractions are identical. In this study we have used immunocytochemical techniques at the electron microscope level to determine whether regional differences exist in the bovine gland with regard to production of individual secretory proteins and whether specialization of product handling occurs at the subcellular level. The technique used is a modification of one previously reported (McLean, J.D., and S.J. Singer. 1970. Proc. Natl. Acad. Sci U.S.A. 69:1771) in which immunocytochemical reagents are applied to thin sections of bovine serum albumin-imbedded tissue and zymogen granule fractions. A double antibody technique was used in which the first step consisted of rabbit F(ab')2 antibovine secretory protein and the detection step consisted of sheep (F(ab')2 antirabbit F(ab')2 conjugated to ferritin. The results showed that all exocrine cells in the gland, and all zymogen granules and Golgi cisternae in each cell, were qualitatively alike with regard to their content of secretory proteins examined (trypsinogen, chymotrypsinogen A, carboxypeptidase A, RNase, and DNase). The data suggest that these secretory proteins are transported through the cisternae of the Golgi complex where they are intermixed before copackaging in zymogen granules; passage through the Golgi complex is apparently obligatory for these (and likely all) secretory proteins, and is independent of extent of glycosylation, e.g., trypsinogen, a nonglycoprotein vs. DNase, a glycoprotein.     

Immunocytochemical expression and localization of protein kinase C in bovine aortic endothelial cells.

Total PKC activity in BAEC incubated for 24 hrs in either 10% serum (FBS) or serum-deprived media (SDM) was similar. However, most of the activity (69%) in the FBS group was detected in the particulate fraction, while it was mainly in the cytosolic fraction (66%) in the SDM group. By confocal microscopy, there was diffuse cytoplasmic localization of the antibodies to the alpha and beta PKC isoforms. gamma PKC was not detected. Treatment of FBS or SDM cells with a phorbol ester resulted in an increase in PKC activity with translocation to the particulate fraction. PKC alpha immunofluorescence redistributed to the perinuclear region whereas PKC beta staining remained mostly cytosolic. Calphostin C, a PKC inhibitor, prevented the phorbol ester-induced increase in PKC activity and translocation.     

Immunocytochemical localization of aquaporin-1 in bovine corneal endothelial cells and keratocytes

For immunocytochemistry, cultured bovine corneal endothelial cells (CBCEC) and bovine corneal cryosections were utilized. Preparations were fixed, permeabilized, and incubated with primary rabbit anti-rat aquaporin 1 (AQP1) antibody followed by rhodamine-conjugated secondary antibody, and were counter-stained with Sytox nuclear acid stain. Confocal microscopy of CBCEC in the x, y, and z planes showed rhodamine fluorescence, indicating the presence of AQP1 antibody localized to the apical and basolateral domains of the plasma membrane, but not to the membranes of intracellular compartments or other subcellular locations. Preabsorption with control antigenic peptide yielded no positive staining. Similar results were obtained using freshly dissected bovine corneas; in addition, these images showed AQP1 distributed to the plasma membranes of keratocytes. No AQP1 staining was seen in corneal epithelium, and no staining was observed in CBCEC layers exposed to AQP3, AQP4, and AQP5 antibodies.     

Immunocytochemical localization of alpha-melanotropin in bovine placenta

The immunocytochemical study of the bovine placenta has demonstrated the presence of cells containing alphamelanotropic hormones, in the decidual epithelium, since the 4th month of pregnancy. The number and the staining intensity of those cells grow up with the placental development. Since the 6th month, we observed the immunoreactional presence of cells in the foetal chorionic epithelium.     

Immunocytochemical localization of beta 1,4 galactosyltransferase in epithelial cells from bovine tissues using monoclonal antibodies.

Post-embedding immunocytochemistry was employed to investigate the distribution of UDP-galactose:N-acetylglucosamine galactosyltransferase (beta 1,4-GT) in epithelial cells from various bovine organs. Several well characterized monoclonal antibodies previously demonstrated to recognize distinct polypeptide epitopes within the primary structure of beta 1,4-GT were applied to thin sections from tissues embedded in Lowicryl K4M, followed by the protein A-gold technique. Immunoreactivity was observed in the Golgi apparatus of epithelial cells from intestine, thymus and trachea. No immunoreactivity was observed in other intracellular structures, including rough endoplasmic reticulum, nuclear envelope and goblet cell mucus droplets. Within the Golgi apparatus, the staining was restricted to several cisternae in the trans region, with most portions of the trans-Golgi network appearing unlabelled. However, in thymic epithelial-reticular cells trans-Golgi network portions resembling classical GERL elements were stained by the antibodies. Thus, although immunoreactivity was subcompartmentalized within the Golgi apparatus in all epithelial cell types examined, the extent of staining within the trans-Golgi network was variable. Immunoreactivity was not detected at the plasma membrane (ecto-galactosyl-transferase), except in the case of a subpopulation of tracheal cells that resemble brush cells. These results suggest that in the epithelial cells examined, the subcompartmental distribution of beta 1,4-GT within the Golgi apparatus is maintained across different types of epithelial cell organization. Moreover, no evidence for a general epithelial cell ecto-galactosyltransferase could be discerned with these reagents.     

Electron probe microanalysis of the subcellular compartments of bovine adrenal chromaffin cells. Comparison of chromaffin granules in situ and in vitro

The elemental and water content of cultured bovine adrenal chromaffin cells and their secretory chromaffin granules have been measured and compared with isolated chromaffin granules using quick freezing, ultracryomicrotomy, and electron microprobe analysis methods. In units of millimole/kilogram dry weight (+/- S.E.) granules in situ contained: P, 523 +/- 32; K+, 124 +/- 9; S, 82 +/- 3; Cl-, 74 +/- 9; Ca2+, 13 +/- 2; Mg2+, 6 +/- 2; and Na+, -2 +/- 2. Following routine isolation in isotonic sucrose buffer, granule K and Cl- had decreased while granule Na+ increased. Cl- exhibited a consistent decrease to 35-40 mmol/kg dry weight. Granule Na+ and K+ concentrations ranged from 43 to 12 mmol/kg and 28 to 60 mmol/kg dry weight, respectively, depending on the Na+ and K+ content of the buffer. Despite the redistribution of monovalent ions, granule Ca2+, granule P, being in the form of ATP, and granule S, being in the form of protein, were not significantly changed. The stability of these elements is consistent with the existence of a stable storage complex for Ca2+, ATP, and protein. Using the granule as an internal standard with a water content of 66%, the water contents of external space, nucleus, cytoplasm, and mitochondria were estimated to be 89, 88, 82, and 70%, respectively. Wet weight concentrations for each element were calculated for granules and cytoplasm from which the transgranular concentration gradients for K+, Cl-, and Na+ were determined. Cl-, a permeant anion, was 2-fold higher in the granule than in the cytoplasm while K+, a slightly permeant cation, had an opposite distribution ratio slightly less than two. Together, the K+ and Cl- data suggest the presence of an inside-positive granule membrane potential of approximately 10-16 mV. The surprising lack of Na+ from the granule matrix suggests a hugh inward gradient for Na+ even though the Na+ content of chromaffin cell cytoplasm is low at 5 mmol/kg water. The lack of an outward Na+ gradient is important in that it indicates that the previously described electroneutral Na+-Ca2+ exchange system, by which isolated granules accumulate Ca2+, does not operate in mature granules in situ. Consequently, if chromaffin granules regulate internal calcium during stimulus secretion coupling, a mechanism other that Na+-Ca2+ exchange is necessary.     

Immunocytochemical localization of NCAM and catecholamine-synthesizing enzymes in rabbit intra- and extra-adrenal chromaffin tissue

Summary The expression of the neural cell adhesion molecule, chromagranin A, and catecholamine-synthesizing enzymes (tyrosine hydroxylase and phenylethanolamineN-methyl transferase) in adrenal medulla and para-aortic bodies (paraganglia) of the adult rabbit, was studied by immunofluorescence. The specificity of the neural cell adhesion molecule antibody employed was demonstrated on rabbit tissue by immunoblotting. Neural cell adhesion molecule was found to be expressed not only by adrenal medullary cells but also by extra-adrenal chromaffin cells present in para-aortic bodies. These paraganglionic cells were as intensely immunolabelled for chromagranin A as adrenal medullary chromaffin cells. They were also labelled for the catecholamine-synthesizing enzymes tested here. However, their levels of the adrenalin-synthesizing enzyme phenylethanolamineN-methyl transferase were lower than those of medullary chromaffin cells.     

Immunocytochemical localization of laminin in rat anterior pituitary cells in vivo and in vitro

Summary The distribution of laminin was investigated by immunocytochemistry in the rat anterior pituitary in vivo and in primary culture. It was localized by immunofluorescence and by immunoperoxidase in the basement membranes of the pituitary in vivo. In addition it was also found inside glandular cells both in vivo and in culture. The number of immunoreactive cells greatly varied depending on the technical approach used. It was always higher in primary cultures than in vivo. At the electron microscope level, a staining was observed on secretory granules, on rough endoplasmic reticulum cisternae as well as on the membrane of some Golgi saccules and vesicles. Such a localization, at the level of subcellular sites involved in the secretory process, suggests that these cells are able to synthesize and to export in vivo as well as in vitro this component of their basement membranes. This work was supported by grants from CNRS (Grant E.R. 89 and ATP “Pharmacologie des Récepteurs des Neuromediateurs”). Part of this work was performed at the EMBL (Heidelberg) during a short stay of C. Tougard (supported by an EMBO short term fellowship). EDITOR'S STATEMENT This paper documents the interesting observation that glandular cells from anterior pituitary contain laminin in their basement membranes and also apparently synthesize and secrete this extracellular matrix component. Gordon H. Sato     

Phosphoglyceride biosynthesis in bovine adrenal chromaffin cells

Cultured adrenal chromaffin cells, representing a virtually homogeneous population of neuronai elements, have been utilized to examine the final enzymes in the formation of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), namely, choline phosphotransferase, ethanolaminephosphotransferase, and the N-methyltransferases in the sequential methylation of PE to PC. Each enzyme has been characterized extensively in terms of substrate requirements, pH optima, detergent and cation effects, and response to inhibitors revealing properties very similar to those in other neural preparations. The respective activities are stable for up to two weeks of adrenal chromaffin cell culture suggesting that this system is a suitable model for examining the relative roles and the regulation of each pathway in PC formation.Abbreviations EPT ethanolaminephosphotransferase - CPT cholinephosphotransferase - NMT N-methyltransferase This work supported by funds provided to the Section of Pediatric Neurology by Texas Children's Hospital.     

Immunocytochemical localization of renin in juxtaglomerular cells

The involvement of various organelles in the synthesis, transport, and packaging of renin in the juxtaglomerular cells of newborn mice has been investigated by immunocytochemistry with the protein A-gold technique. Highly specific rabbit antibodies against mouse submandibular renin were used. Mild fixation and embedding in glycol methacrylate allowed enough sensitivity to identify a steep gradient of labeling from rough endoplasmic reticulum to Golgi complex to secretory granules. Routine fixation and embedding in Epon produced labeling differentials that allowed delineation of hitherto ill-defined types of secretory granules and vacuoles. The classical pattern of synthesis, transport, and packaging of secretory proteins involves the rough endoplasmic reticulum and Golgi complex and seems to apply to renin secretion. Immunoreactive renin is packaged as rhomboid crystals at the trans face of the Golgi complex. The limiting membrane of these rhomboids fuses to form coalescing protogranules where the crystals eventually yield their individuality maturing into secretory granules. Vacuoles containing a flocculent material, with or without a dense core, show significant immunocytochemical labeling. These vacuoles are not associated with the Golgi complex but occupy cytoplasmic areas well endowed with rough endoplasmic reticulum. As judged from their morphological features and their immunoreactivity, the vacuoles do not seem to follow the sequence of events typical of protogranules and coalescing protogranules. They possibly represent a parallel pathway of renin synthesis and transport, involving the nuclear envelope and bypassing the Golgi complex.     

Immunocytochemical localization of prostaglandin endoperoxide synthase in the bovine intestine

The localization of prostaglandin (PG) endoperoxide synthase in bovine intestine was examined immunocytochemically with polyclonal antibody raised against PG endoperoxide synthase purified from bovine seminal glands. The most intense positive staining reaction for the enzyme was present in mast cells. Mast cells were found to be widely distributed in the intestinal wall, and were particularly numerous in the lamina propria. Most of the mast cells in the lamina propria of the intestinal villi were elongated and oriented with their long axis parallel to the plane of the absorptive epithelium. In whole mount preparations of jejunal villi, mast cells were seen to form a two-dimensional network in the lamina propria. In addition to mast cells, smooth muscle cells of the inner circular muscle layer and muscularis mucosae, nerve cells and fibers, endothelial cells of arterioles, and serosal epithelial cells also showed faint to moderate staining for the enzyme. These results suggested that mast cells are the major source of PGs in the bovine intestinal wall. The characteristic arrangement of mast cells in the intestinal villi may be related to their functions in this portion of the bovine intestine.     

Adenosine transport in bovine chromaffin cells in culture

Bovine adrenal chromaffin cells in culture have a high capacity and affinity for adenosine uptake with Vmax = 14 +/- 2.4 pmol/10(6) cells/min (133 pmol/mg of protein/min) and Km = 1 +/- 0.2 microM. Transport studies, at short time periods, in recently isolated chromaffin cells have Vmax = 15 pmol/10(6) cells/min and Km = 1.1 microM in ATP-depleted cells. Endogenous levels of the various purine nucleosides and bases were determined by high pressure liquid chromatography, with adenosine (3 +/- 1 nmol/10(6) cells), inosine (5.3 +/- 1.2 nmol/10(6) cells), and hypoxanthine (2.1 +/- 0.8 nmol/10(6) cells) being the purine metabolites found in the highest concentration. Taking into account the intracellular water, endogenous levels of 2.1, 3.8, and 1.5 mM, respectively, were obtained. Radioactively labeled adenosine inside the cell underwent enzymatic transformations, producing inosine, hypoxanthine, xanthine, and nucleotides, with their appearance and distribution being a function of the incubation time. When nicotine was used as a secretagogue, the adenosine transformed into the nucleotide pool was released, reaching 18 +/- 8% of the total adenosine found in the nucleotides. Dipyridamole, extensively used clinically, was a strong inhibitor for the adenosine uptake into these cells, with Ki = 5 +/- 0.5 nM and noncompetitive kinetically.     

Immunocytochemical localization of tryptophanyl-tRNA-synthetase in a line of bovine kidney cells and in sublines with elevated enzyme levels

Intracellular localization of tryptophanyl-tRNA synthetase (E. C. 6.1.1.2) has been studied immunocytochemically using monospecific antibodies in cultured bovine kidney cells (strain MDBK) and in substrains with elevated enzyme levels. Both light and electron microscopy were used and native or detergent-treated cells were examined. The product of cytochemical reaction was revealed on free polyribosomes, polyribosomes attached to membranes of granular endoplasmic reticulum, on cytofilaments and in the nucleus as well.     

Calcium-dependent transferrin receptor recycling in bovine chromaffin cells

The release of regulated secretory granules is known to be calcium dependent. To examine the Ca²⁺-dependence of other exocytic fusion events, transferrin recycling in bovine chromaffin cells was examined. Internalised ¹²⁵I-transferrin was released constitutively from cells with a half-time of about 7 min. Secretagogues that triggered catecholamine secretion doubled the rate of ¹²⁵I-transferrin release, the time courses of the two triggered secretory responses being similar. The triggered ¹²⁵I-transferrin release came from recycling endosomes rather than from sorting endosomes or a triggered secretory vesicle pool. Triggered ¹²⁵I-transferrin release, like catecholamine secretion from the same cells, was calcium dependent but the affinities for calcium were very different. The extracellular calcium concentrations that gave rise to half-maximal evoked secretion were 0.1 m m for ¹²⁵I-transferrin and 1.0 m m for catecholamine, and the intracellular concentrations were 0.1 μ m and 1 μ m , respectively. There was significant ¹²⁵I-transferrin recycling in the virtual absence of intracellular Ca²⁺, but the rate increased when Ca²⁺ was raised above 1 n m , and peaked at 1 μ m when the rate had doubled. Botulinum toxin type D blocked both transferrin recycling and catecholamine secretion. These results indicate that a major component of the vesicular transport required for the constitutive recycling of transferrin in quiescent cells is calcium dependent and thus under physiological control, and also that some of the molecular machinery involved in transferrin recycling/fusion processes is shared with that for triggered neurosecretion.