Immunohistochemical localization of constitutive and inducible cyclo-oxygenases in rat uterus during the oestrous cycle and pregnancy

The uterus is a rich source of eicosanoids synthesized from arachidonic acid metabolism through the cyclo-oxygenase pathway. Two isoforms of cyclo-oxygenase, constitutive (COX-I) and inducible (COX-II) enzyme, have been reported. In the present study, we have immunohistochemically mapped the distribution of both COX-I and COX-II during various physiological states of the rat uterus. Uterine tissue was collected from female rats (a) during different stages of the oestrous cycle, (b) on days 1, 4, 8 and 18 of gestation, (c) after spontaneous delivery and (d) post partum, and fixed in Bouin's fixative. After paraffin wax embedding, 5-m-thick sections were immunohistochemically stained by the ABC technique. Observation of the stained sections under the light microscope revealed that, in non-pregnant rat uterus, both COX-I and COX-II were abundantly expressed in the endometrium, with minimal staining observed in the myometrium. Staining was more prominent in epithelial cells than in stromal cells. The intensity of staining in epithelial cells was highest at pro-oestrus and oestrus and lowest at dioestrus. In pregnant rats, although the expression of both COX-I and COX-II was localized primarily to the endometrium with very little staining in the myometrium on day 1 of gestation, both of these enzymes were also apparent in myometrial cells by day 4 of gestation. The staining intensity of endometrial and myometrial cells increased further with the progression of gestation, being maximal at the time of spontaneous delivery. During the post-partum period, however, the staining intensity for both of the enzymes in endometrium and myometrium was decreased. Thus, our studies show that the expression of cyclo-oxygenases in various uterine cells vary with the oestrous cycle and with pregnancy. Furthermore, prominent increases in the expression of cyclo-oxygenases in the myometrium during pregnancy and parturition imply that the cyclo-oxygenase system in the myometrium may play a major role in modulating uterine contractility during pregnancy and labour. © 1998 Chapman & Hall     

Transforming growth factor beta1 involvement in the conversion of fibroblasts to smooth muscle cells in the rabbit bladder serosa

In an attempt to identify the growth factors or cytokines involved in the serosal thickening that occurs in rabbit bladder subjected to partial outflow obstruction, the following growth factors – transforming growth factor 1, platelet-derived growth factor, epidermal growth factor, granulocyte colony-stimulating factor and granulocyte–monocyte colony-stimulating factor – were delivered separately onto the serosal surface of the intact bladder via osmotic minipumps. The proliferative/differentiative cellular response of the rabbit bladder wall was evaluated by bromodeoxyuridine incorporation and immunofluorescence staining with a panel of monoclonal antibodies to cytoskeletal proteins (desmin, vimentin, keratins 8 and 18 and non-muscle myosin) and to smooth muscle (-actin, myosin and SM22) proteins. Administration of the transforming growth factor, but not of the other growth factors/cytokines, was effective in inducing serosal thickening. Accumulating cells in this tissue were identified as myofibroblasts, i.e. cells showing a mixed fibroblast–smooth muscle cell differentiation profile. The phenotypic pattern of myofibroblasts changed in a time-dependent manner: 21 days after the growth factor delivery, small bundles of smooth muscle cells were found admixed with myofibroblasts, as occurs in the obstructed bladder. These ectopic muscle structures displayed a variable proliferating activity and expressed an immature smooth muscle cell phenotype. The complete cellular conversion to smooth muscle cells was not achieved if transforming growth factor 1 was delivered to fibroblasts of subcutaneous tissue. These findings suggest a tissue-specific role for this growth factor in the cellular conversion from myofibroblast to smooth muscle cells. © 1998 Chapman & Hall     

Hints of a functional connection between the neuropeptidergic innervation of arteriovenous anastomoses and the appearance of epithelioid cells in the rabbit ear

Peripheral blood flow can be regulated by specialized vessel segments, the arteriovenous anastomoses. Their wall consists of a relatively thick layer of smooth muscle cells and so-called epithelioid cells. The epithelioid cell is a specialized myogenic cell phenotype expressing nitric oxide synthase. We studied the innervation of the different segments of arteriovenous anastomoses in the rabbit ear using antisera against neuropeptide Y, tyrosine hydroxylase, calcitonin gene-related peptide and substance P, as well as neuron-specific enolase, calbindin D and neurotubulin. The participation was especially examined of neuropeptidergic innervation and a possible morphological connection to the occurrence of epithelioid cells and a paracrine function. The NADPH diaphorase reaction and -smooth muscle actin immunoelectron microscopy served to distinguish epithelioid cells from smooth muscle cells. Using conventional fluorescence microscopy and confocal laser scanning microscopy, we found the most dense innervation pattern of pan-neuronal markers (neurotubulin, neuron-specific enolase), tyrosine hydroxylase-immunor eactive nerve fibres and neuro-peptidergic nerve fibres (neuropeptide Y, calcitonin gene-related peptide, substance P) around the intermediate segment in arteriovenous anastomoses, whereas the venous segment was barely marked. Single nerve fibres penetrated into the medial layer and reached the epithelioid cells. Using immunoelectron microscopy, we found intercellular contacts between epithelioid cells, but not the gap junction protein connexin 43. Here, we report for the first time a correlation of the innervation pattern with epithelioid cell type in arteriovenous anastomoses. Our findings suggest that epithelioid cells of the arteriovenous anastomoses are controlled by a dense network of neuropeptidergic nerve fibres in functional connection to their paracrine role as a nitric oxide producer. © 1998 Chapman & Hall     

Production of monoclonal antibody against histamine and its application to immunohistochemical study in the stomach

A monoclonal antibody against histamine has been produced. A histamine–haemocyanin conjugate prepared using 1-ethyl-3-(3-dimethylami nopropyl) carbodiimide as a coupling agent was used for immunizing mice. Immunized mice were sacrificed to prepare monoclonal antibody using a hybridoma technique. On immunospot assay, the hybridoma culture supernatant containing a monoclonal antibody was capable of detecting 50 pmol of histamine. Using this antibody, we examined the cellular localization of histamine-like immunoreactivity in the stomach of normal or -fluoromethylhistidine-treated rats and mice. Immunoreactive cells were abundant in the gastric mucosal layer. These positive cells were often located in the basal half of the fundic gland but were rare in the pyloric gland. The cells, small or medium in size, spindle or cone in shape, were intermingled with immunonegative epithelial cells. In the cytoplasm of the positive cells, granular reaction products were densely deposited. In addition, a few positive cells, identified as mast cells by Toluidine Blue staining, were distributed mainly in the submucosal and muscular layer. The antibody preabsorbed with 10 mm histamine gave no positive immunostaining. For pharmacological study, some rats were injected six times with -fluoromethylhistidine every 8 h. In these rats, positive cells except mast cells were no longer detected. In conclusion, the monoclonal antibody produced appears to be highly specific for histamine. Its application in immunohistochemistry should provide a powerful tool for analysing the roles of histamine in enterochromaffin-like or mast cells in the stomach. © 1998 Chapman & Hall     

Characterization of glycoconjugates of guinea pig seminal vesicle by lectin histochemistry

In the present study, the expression of glycoconjugates in the guinea pig seminal vesicle was localized and partially characterized by lectin histochemistry using a battery of 30 different lectins specific for different carbohydrate residues. The results indicate that the glandular epithelium of the guinea pig seminal vesicle exhibits complex glycoconjugates rich in Man, -GlcNAc, -Gal, /-GalNAc, Fuc and complex NeuAc(2,6)Gal/GalNAc residues, as shown by its positive reactions to most lectins used. The Golgi region of the luminal secretory epithelial cells expresses a complex glycoconjugate pattern, as shown by its strong reactions to Man-(PSA, GNA), -GlcNAc-(S-WGA, PWA, DSA, UDA), -Gal- (RCA-I and -II), /-GalNAc-(SBA, Jac, VVA, BPA) and complex NeuAc-(SNA) specific lectins, indicating that the secretory epithelial cells are active in glycosylation and secretion process. It was also shown in the present study that the basal and luminal epithelial cells are different in their glycoconjugates. The basal epithelial cells are rich in NeuAc(2,3)Gal residues as they are stained specifically by MAA. The fibroblasts in the epithelial-smooth muscle interface and the smooth muscle cells close to the glandular epithelium are shown to express more glycoconjugates as they are stained intensely by GS-I-B4, GS-II and SBA. However, their role in the epithelial-stromal interaction in the seminal vesicle remains to be elucidated. In summary, the present study reports for the first time on the lectin binding patterns of the guinea pig seminal vesicle, and the results show that the seminal vesicle epithelium elaborates and secretes glycoconjugates in a complex pattern. Some of the lectins might be useful as histochemical markers for the secretory activity and specific structural components in the guinea pig seminal vesicle. © 1998 Chapman & Hall     

Immunohistochemical localization of tonin in rat salivary glands and kidney

Summary As a part of a microfluorometric investigation of the nucleoproteins of nuclei whose chromatin displays varying degrees of condensation, a comparison was made of mouse small thymocyte and hepatocyte nuclei stained with the acidic dye, brilliant sulfaflavine, at pH 2.8. These estimates of total protein content were compared with measurements obtained in similarly stained nuclei after extraction either with 0.4 N H₂SO₄ to remove all histones or with 0.35 M NaCl to remove nucleoplasmic proteins and some loosely bound non-histone chromosomal proteins. Treatment with 5% TCA at 60°C was used to remove nucleic acids and to reverse the effects of formaldehyde fixation. In all instances, the fluorescence of 2c hepatocyte nuclei greatly exceeded that of similarly treated thymocyte nuclei. While extraction with 0.4 N H₂SO₄ resulted in reductions of as much as 75% of the total fluorescence of small thymocyte nuclei, the losses of fluorescence in 2c hepatocyte nuclei amounted to only 20–30%. Nevertheless, the absolute values of fluorescence lost in both types of nuclei were very similar. After extraction in 0.35 M NaCl, thymocyte nuclei displayed slightly greater fluorescence than control thymocyte nuclei, while the total fluorescence of hepatocyte nuclei declined. In hepatocyte nuclei extracted with TCA, with and without treatment with 0.35 M NaCl, two populations of diploid nuclei were apparent: one corresponding to parenchymal cell nuclei and the other comprised of non-parenchymal cell nuclei. Only single diploid populations were visible in acid-extracted material. The ratios of 4c2c, 8c4c, and 8c2c hepatocyte nuclei in control, acid-extracted, and NaCl-extracted groups were generally lower than the expected 224 values. These results indicate that total nuclear histones may be estimated microfluorometrically by computing the difference between acid-extracted and unextracted preparations treated in otherwise equivalent ways. In addition, despite very similar absolute losses of fluorescence after removal of histones in thymocyte and 2c hepatocyte nuclei, the proportion of total protein ascribable to histones is much greater in thymocyte nuclei than in 2c hepatocyte nuclei — or, conversely, the percentage of total protein attributable to non-histone proteins is much greater in 2c hepatocyte nuclei than in thymocyte nuclei.     

Immunohistochemical localization of tonin in rat salivary glands and kidney

Tonin has been localized in salivary glands and kidney by the indirect immunofluorescence technique of Coons and by the unlabeled antibody technique of Sternberger. Both techniques gave identical results. Immunoreactive tonin was localized in the cytoplasm of granular convoluted tubular cells and on the apical surface of striated duct cells and collecting duct cells of the submandibular gland. In the parotid and sublingual glands, which lack granular cells, tonin was only found on the apical surface of striated duct and collecting duct cells. In the kidney, immunoreactive tonin was found only associated with cells of the distal convoluted tubules. After fixation with Bouin fluid or with ethanol, tonin was found not only on the apical surface of the cells but also in the apical and perinuclear cytoplasm. This cytoplasmic staining has been attributed to artefactual diffusion since, after fixation with formol-picric acid, the enzyme could only be localized on the apical surface of the tubular cells.     

Localization of neurotensin binding sites in rat kidney

[3H]Neurotensin ([3H]NT) appears to bind specifically to a single class of sites in slide-mounted rat kidney sections (KD = 8.3 nM; Bmax = 31.6 fmol/mg tissue). Bound [3H]NT can be displaced by nonradioactive NT and a series of its fragments and analogues with relative potencies that correlate well (r = 0.91; p less than 0.005) to their potencies in the rat stomach strip bioassay. These results suggest that NT receptors are similar in both systems. However, they are probably slightly different from those present in the guinea pig atria (r = 0.78; p less than 0.1). We visualized these sites by using the tritium-sensitive LKB film technique analysed by computerized densitometry. [3H]NT binding sites are highly concentrated in the renal cortex while low levels are observed in the renal medulla. The possible physiological and/or pathophysiological significances of the presence of [3H]NT binding sites in the kidney are discussed.     

Immunohistochemical localization of sodium-dependent l-ascorbic acid transporter 1 protein in rat kidney

Recently, two l-ascorbic acid transporters were identified; sodium-dependent vitamin C transporter (SVCT) 1 and SVCT2. The previous study suggested that SVCT protein might be present on the apical membrane in the straight segment (S3) of proximal tubule. In the present study, SVCT1 immunoreactivity (IR) was observed in the brush border of proximal straight tubules in the medullary ray of renal cortex and the outer stripe of outer medulla, while SVCT2 IR was not localized in any region of the kidney. Since the mechanism of VC reabsorption in the kidney has not been fully elucidated up to the present time, it is meaningful to demonstrate the exact cellular distribution of SVCT protein in the kidney.     

Immunohistochemical localization of 11-hydroxysteroid dehydrogenase in rat kidney with monoclonal antibody

Monoclonal antibody (MAb) against 11-hydroxysteroid dehydrogenase (11-HSD) has been raised by immunization of female balb/c mice. 11-HSD from solubilized rat renal microsomal protein could be bound in a modified ELISA using antimouse IgG and MAb against 11-HSD. On Western blots of solubilized rat renal microsomes the MAb recognized a single protein band of an approximate molecular weight of 35 kD. Immunohistochemical staining of rat renal tissue with the above MAb and the APAAP staining technique displayed a heterogenous reginal and subcellular distribution: glomeruli and arterioles were practically devoid of specific staining, as were epithelial cells in inner and outer medulla. Intense immunostaining was observed in PCT and particularly in PST, appearing granular with highest density around the nuclei. Here the enzyme bound to intracellular membranes may exert an autocrine function such as signal inactivation. In contrast to cortex, staining of interstitial cells was observed in renal medulla. The latter localization is compatible with the concept of a paracrine function of 11-HSD which might prevent corticosterone from gaining access to collecting duct cells.     

Immunohistochemical localization of prolactin-binding sites in R3327 rat prostatic cancer cells.

Light microscope immunohistochemistry was used to test for and to localize prolactin-binding sites in the transplantable R3327 rat prostatic carcinoma. Fixed tissue sections of the tumor were first incubated with vehicle or varying concentrations of highly purified NIAMDD rat prolactin and then exposed to an immunoperoxidase staining sequence. Prolactin produced dose-related, immunospecific, staining in the cytoplasm of some neoplastic cells, indicative of intracellular prolactin-binding sites (IPBS). While cells with IPBS have been demonstrated in both differentiated and undifferentiated regions of this cancer, a role for prolactin in prostatic neoplasia, be it stimulatory or inhibitory, remains to be elucidated.     

Immunocytochemical localization of cathepsin D in lysosomes of cortical collecting tubule cells of the rat kidney

Immunocytochemical localization of cathepsin D in rat renal tubules was investigated by means of indirect immunoenzyme and protein A--gold techniques. By light microscopy, fine granular staining was seen in the mesangial cells of glomeruli. Heavy reaction deposits were present in the cortical tubular segments and some of the medullary collecting tubules. The proximal tubules contained a few positive granules. Other segments were negative for cathepsin D. By electron microscopy, gold particles representing the antigenic sites for cathepsin D were present in cytoplasmic granules and multivesicular bodies of the segment of the cortical collecting tubule. These cytoplasmic granules were presumed to be digestive vacuoles (secondary lysosomes) from their morphological profile. The proximal tubule cells contained the very weakly labeled secondary lysosomes. No specific labeling was noted in other segments of the nephron. Control experiments confirmed the specificity of the immunostaining. Quantitative analysis of the labeling density in each subcellular compartment also confirmed that the main subcellular sites for cathepsin D are the secondary lysosomes and multivesicular bodies. The labeling density in these granules of the lysosomal system varied widely with the individual granules, suggesting that there is a considerable heterogeneity of enzyme content among the granules of the lysosomal system. The prominent presence of cathepsin D in the cortical collecting tubule suggests a certain segment-specific function of this proteinase.     

Ultrastructural localization of glucose-6-phosphatase activity in proximal convoluted tubule cells of rat kidney

Summary The ultrastructural localization of glucose 6-phosphatase activity was investigated in the proximal convoluted tubule cells of the rat kidney. The reaction product for the enzyme activity was present in the endoplasmic reticulum and nuclear envelope, as reported for the hepatic enzyme and others, but was absent from the brush border, plasma membrane and other organelles. The metabolic significance of the association of this enzyme with the endoplasmic reticulum and the role of the enzyme in the active reabsorption and transport of glucose in the renal tubules are discussed.     

In vitro autoradiographic localization of [I]-angiotensin II binding sites in the rat and dog kidney

Light microscopic autoradiographic techniques have been utilized to demonstrate specific regions of the rat and dog kidney where angiotensin II receptors exist. Slide mounted tissue sections were labeled with [¹²⁵I]-angiotensin II using conditions which provided for highly specific binding. These angiotensin II binding sites were localized to several distinct renal structures. In the renal cortex, angiotensin II binding sites were found concentrated in all parts of the glomeruli including the vascular components, the macula densa and the juxtaglomerular apparatus. Angiotensin II binding in the medulla was more diffusely associated with the vasa recta, and to a lesser extent, the thick ascending segment of the loop of Henle. Binding sites specific for angiotensin II were also found in the smooth muscle laminae of the ureter. Scatchard analysis of the binding kinetics allowed the demonstration of two subpopulations of binding sites which differ slightly in their affinities for [¹²⁵I]-angiotensin II. These subpopulations appear to be associated with distinct components of the renal structure.     

Distribution of IGF-I mRNA and IGF-I binding sites in the rat kidney.

In the present study we have investigated the distribution of IGF-I mRNA and IGF-I binding sites in the rat kidney. The distribution of IGF-I mRNA was investigated using a simple and sensitive non-radioactive in situ hybridisation technique based on probe labelling with digoxigenin labelled-UTP followed by detection with conventional immunocytochemical techniques. IGF-I mRNA was found predominantly in medullary collecting ducts and sparsely in cortical collecting duct cells. In addition IGF-I mRNA was expressed in scattered proximal tubular cells in the cortex and in cells confined to the glomerular tuft. IGF-I binding sites were studied using radiolabelled IGF-I and conventional autoradiographical techniques on tissue sections. It was found that IGF-I binding sites were widely distributed throughout the entire kidney and that the specific binding was highest in the inner medulla. These findings add further complexity to the understanding of IGF-I production and action on renal structures.     

A post-embedding avidin-biotin peroxidase system to demonstrate the light and electron microscopic localization of lectin binding sites in rat kidney tubules

Summary A post-embedding method for the light and electron microscopic demonstration of lectin binding sites in rat kidney tubules is described. The use of biotinylated lectins, followed by treatment with avidin peroxidase and the DAB—H₂O₂ sequence, produced intense staining of acrylic sections at the electron microscope level: brush borders and associated structures, cytoplasmic granules, basal infoldings and basement membrane—plasmalemmal interfaces of proximal tubules bound erythrophytohaemagglutinin, while distal tubules were mainly unstained. At the light microscope level, epoxy resin sections showed a similar staining pattern after etching, as did acrylic resin sections after intensification of the final reaction product. The binding of wheatgerm agglutinin to cytoplasmic granules and brush border structures in the proximal tubules was abolished, at both the light and electron microscope levels, by the competing sugar tri-N—acetylchitotriose. Epoxy resin ultrathin sections required etching before staining was achieved in the electron microscope, and results were far inferior to those obtained with acrylic resin. This method allows rapid and inexpensive screening of large numbers of lectins, if required, at both the light and electron microscope levels, using reagents that are stable for long periods of time.     

Immunohistochemical localization of amylin in rat brainstem

Immunohistochemical studies were conducted on rat brainstem using a specific polyclonal antiserum against the COOH-terminal (25-37) of human amylin. Amylin-immunoreactive cell bodies were observed in the vestibular, cochlear, trapezoid, and inner cerebellar nuclei and in the mesencephalic nucleus of trigeminal nerve. Positive cell bodies were also found in lateral, gigantocellular and magnocellular reticular nuclei. Numerous amylin-immunoreactive nerve fibers were shown in the trigeminal spinal tract, in the solitary area and in the area postrema. Amylin-immunoreactive cell bodies were often surrounded by a network of tyrosine hydroxylase-immunoreactive nerve fibers. These results provide morphologic evidence that amylin may play a role in some discrete sensory functions.