Immunochemistry of prostaglandin endoperoxide-forming cyclooxygenases: the detection of the cyclooxygenases in rat, rabbit, and guinea pig kidneys by immunofluorescence.

Rabbit antiserum has been prepared against the prostaglandin endoperoxide-forming cyclooxygenase (EC 1.14.99.1) purified from sheep vesicular glands. Ouchterlony double diffusion and immunoelectrophoretic analyses indicate that the anti-cyclooxygenase serum is monospecific for the enzyme. The anti-cyclooxygenase serum reacts with both active and inactivated forms of the sheep vesicular gland (SVG) cyclooxygenase. Furthermore, the immune serum precipitates solubilized microsomal cyclooxygenases from each of six other tissues examined, including bovine seminal vesicle, rabbit kidney medulla, guinea pig lung, dog spleen, sheep uterus, and human platelets. Anti-SVG cyclooxygenase serum was used in combination with fluorescein isothiocyanate )FITC)-labeled goat anti-rabbit IgG to detect cyclooxygenases in cryostat sections from rat, rabbit and guinea pig kidneys by immunofluorescence. Highly prominent fluorescence was associated only with the epithelial cells lining the collecting ducts in rabbit and guinea pig kidneys, and except for the nucleus, was uniformly distributed within the interior of these cells. In rat kidney, fluorescence was detected not only in collecting tubules but also in the interstitial cells of the renal papilla. Our results are consistent with the emerging hypothesis that PGE2 produced intrarenally plays a physiological role in natriuresis.     

Effect of anti-pig zona pellucida serum on fertilization in several mammals

The effect of goat antiserum against isolated pig zonae pellucidae on fertilization in vivo was examined in the pig, cow, sheep, rabbit, rat, and mouse. As shown by indirect immunofluorescence, anti-pig zona serum reacted strongly with the zonae of pig, cow, sheep, and rabbit, but the reaction with the zonae of mouse and rat was weak. Passive immunization with anti-pig zona serum significantly, or completely, inhibited fertilization in all species. However, inhibition of fertilization was more pronounced in the pig, cow, sheep, rabbit, and mouse than in the rat. Inhibition of fertilization in the rabbit was also observed after passive immunization with antiserum absorbed with rabbit liver and kidney. All of the zonae recovered from the pig, cow, sheep, rat, and mouse after passive immunization with anti-pig zona serum exhibited strong fluorescence, regardless of the incidence of fertilization. It was concluded that the pig and other mammalian zonae pellucidae tested have tissue-specific antigens.     

Comparative studies of the protein composition of red blood cell membranes from eight mammalian species

The polypeptide pattern of red blood cell (RBC) membranes from cow, sheep, horse, rabbit, guinea pig, rat, mouse, analyzed by polyacrylamide gel electrophoresis, was compared to human RBC counterpart. Some qualitative and quantitative differences were noted. Among the high molecular weight components the bands 2.1- 2.3 appeared slightly decreased in rabbit and rat and increased in sheep RBC membranes. Band 3 appeared to have a higher molecular weight in the cow, guinea pig and mouse RBCs, and a lower molecular weight in the sheep RBCs. Band 4.1 from the RBC membranes of cow, sheep, rabbit and guinea pig was splitted into two sub-bands, while band 4.2 overlapped with band 4.1 in horse and guinea pig RBC membranes. There are marked differences in the number and position of bands in the 4.5 region, while band 4.9 is present in higher amounts in horse, rabbit and guinea pig RBC membranes. Band 6 (glyceraldehyde 3-phosphate dehydrogenase) was undetectable in horse, rat and mouse RBC membranes and was decreased in sheep, rabbit and guinea pig. There are also major differences in the region of band 7 and below ("post-7"). Band 8 was undetectable in horse, cow and guinea pig, and was in higher amounts in rat. A band corresponding to a molecular weight of about 22 kD in the "post-8" region was present only in guinea pig RBC membranes.     

Alanine: glyoxylate aminotransferase 1 is present in the peroxisomes of guinea pig kidney

The subcellular distribution of alanine: glyoxylate aminotransferase 1 in guinea pig and rabbit kidneys was examined by centrifugation in a sucrose density gradient. The enzyme was located in the peroxisomes of guinea pig kidney and cross-reactive with the antibody against rat liver alanine: glyoxylate aminotransferase 1. This is the first report on the presence of the enzyme in the peroxisomes of mammalian kidney. The enzyme was found to be located in the mitochondria but not in the peroxisomes in rabbit kidney.     

Immunocytochemical localization of the prostaglandin-forming cyclooxygenase in the cerebellar cortex

An indirect immunocytofluorescence technique was used to examine the distribution of the prostaglandin-forming cyclooxygenase in the cerebellar cortex of the pig, guinea pig, rat, mouse, cow, rabbit and sheep. Cyclooxygenase antigenicity was detected (a) in the cell bodies of Bergman glial cells in the Purkinje cell layer of the porcine, ovine and bovine cerebellar cortex; (b) in small arterioles throughout the cerebellar cortex in the sheep and cow; and (c) in the endothelial cells of large arteries in all the species examined. No cyclooxygenase-positive staining was apparent in neuronal cell bodies of granule, basket, stellate or Purkinje cells. Our results establish that prostaglandin endoperoxides can be synthesized by the arterial vasculature and at least certain glial cells in the central nervous system.     

Identification of thymus-dependent areas in frozen sections of guinea pig lymphatic tissue by adherence of rabbit erythrocytes

Untreated rabbit erythrocytes adhere to thymus-dependent areas of guinea pig lymphatic tissues as shown with frozen sections. The adherence reaction is temperature dependent. Optimal results were obtained by incubation of the tissue section with the erythrocytes at temperatures between 0 ° and 4 °C. At 37 °C no adherence of erythrocytes was observed. Out of other erythrocytes tested (human, sheep, chicken, rat, mouse) only rat and mouse cells showed weak adherence to guinea pig thymus sections.     

Effects of heterologous sera on fertilized rabbit ova

Undiluted blood serum of various species was used to culture two-celled rabbit ova for 24 hours. It was found that there is an ovocidal factor present in the serum of man, sheep, cattle, goat, and fowl. The factor is lethal rather than inhibitory; exposure to it for 10 minutes will cause the death of the ova. This factor is unstable, thermolabile (destroyed at 55 degrees C. in 30 minutes), and of large molecular size. The strength or concentration of this factor varies according to the origin of the serum, increasing in the order man, sheep, cattle, goat, fowl. The blood serum of rabbit, horse, dog, guinea pig, rat, and pig contains no ovocidal factor against rabbit ova. The ovocidal factor differs from the spermicidal factor, which is present in all the sera of the different species studied with rabbit spermatozoa. Immunization of the guinea pig against rabbit ova is possible. Normal development of young rabbits was obtained by transplantation of ova cultured in the heated or normal serum of other species after 24 hours.     

Immunocytochemical localization of the prostaglandin-forming cyclooxygenase in the cerebellar cortex

An indirect immunocytofluorescence technique was used to examine the distribution of the prostaglandin-forming cyclooxygenase in the cerebellar cortex of the pig, guinea, rat, mouse, cow, rabbit and sheep. Cyclooxygenase antigenicity was detected (a) in the cell bodies of Bergman glial cells in the Purkinje cell layer of the porcine, ovine and bovine cerebellar cortex; (b) in small arterioles throughout the cerebellar cortex in the sheep and cow; and (c) in the endothelial cells of large arteries in all the species examined. No cyclooxygenase-positive staining was apparent in neuronal cell bodies of granule, basket, stellate or Purkinje cells. Our results establish that prostaglandin endoperoxides can be synthesized by the arterial vasculature and at least certain glial cells in the central nervous system.     

Cyto-immunological studies of guinea pig sperm antigens

Summary Antigenic localization in guinea pig epididymal sperm and testicular imprints as well as in viable, motile guinea pig epididymal sperm was studied by means of fluorescent labelled antibody techniques. Globulins from rabbits and chickens immunized with guinea pig epididymal sperm were used in the direct procedure while sera from sheep and fowl injected with rabbit globulins were used in the indirect procedure. The main findings were: 1) spermatozoa from the distal portion of the epididymis displayed brilliant fluorescent acrosomes and less intensely stained midpieces and principal pieces when treated as dried smears in both the direct and indirect methods; 2) testicular spermatozoa were similarly stained but whereas in epididymal spermatozoa the whole acrosome stained intensely, the testicular spermatozoal acrosome displayed intense fluorescence of the inner acrosome; 3) protoplasmic droplets fluoresced strongly; 4) cross-reactivity was observed between human and guinea pig sperm but not between rat and guinea pig sperm, indicating an antigenic relationship between human and guinea pig but not between guinea pig and rat; 5) treatment of viable, motile guinea pig spermatozoa with fluorescent globulins resulted in agglutination and immobilization as well as formation of antigen-antibody aggregates adherent to the cell membrane of the head, midpiece and principal piece; the formation of such fluorescent aggregates in the medium surrounding the treated motile sperm was indicative of leaching of antigenic material from the sperm cells.This investigation was supported by funds from United States Public Health Service grant HE-05798-03, The Ford Foundation and National Science Foundation.     

Immunocytochemical localization of a renal glycoprotein (gp CD I) synthesized by cultured collecting duct cells

Summary A sulfated, proline-rich glycoprotein (gp_CDI, apparent molecular weight 200,000 in column chromatography and 150,000 in SDS-PAGE) was isolated from cultured renal collecting duct epithelium by centrifugation, Triton X100 extraction and DEAE-cellulose ion exchange chromatography. A DEAE-cellulose ion exchange chromatography fraction with the enriched gp_CDI was used for immunization of guinea pigs. The antiserum was prepared for antigen localization by indirect immunofluorescence in collecting duct cell cultures and in tissue sections of neonatal and adult rabbit kidneys. In the cultured collecting duct epithelium, antibody staining of the epithelium and structures of the extracellular matrix was age dependent. Cultures of dedifferentiated collecting duct monolayers revealed positive reaction in the cytoplasm. In neonatal and adult rabbit kidneys, the antibody was localized in the entire collecting duct system but not in the collecting duct ampullae of the newborn kidney. Staining of the cytoplasm was found only in medullary collecting ducts of the neonatal kidney; other portions revealed staining mostly at the basal circumference of the tubule and at the luminal cell borders. Apart from collecting ducts, no other tubular segments were reactive. The cortical and the medullary interstitium contained fluorescent fibres which were concentrated around vascular structures. A possible relation between gp_CDI and collagenous compounds is discussed. Bowman's capsule reacted positively, whereas staining of the mesangial matrix was weak. The localization of the antigen, as revealed by indirect immunofluorescence, suggests that gp_CDI occurs both in intracellular and extracellular (interstitial) location. Two main points are emphasized: Firstly, gp_CDI is considered an important constituent in different stages of collecting duct development, and secondly, the staining pattern of the antibody varies with the different portions of both young and adult kidney collecting ducts; this staining heterogeneity may correspond with the known regional differences of collecting duct functions.     

Increased renal tubular synthesis of prostaglandins in the rabbit kidney in response to ureteral obstruction

The hydronephrotic rabbit kidney exhibits elevated basal prostaglandin synthesis and supersensitivity to peptide stimulation of vascular prostaglandin and thromboxane formation. In this study the distribution of the prostaglandin-forming cyclooxygenase in hydronephrotic and contralateral rabbit kidneys following one and four day ureteral obstructions was compared using immunohistofluorescence. No alterations were detected in the distribution or intensity of cyclooxygenase-positive fluorescence in the renal vasculature in response to ureteral obstructions. However, two significant differences were noted between hydronephrotic and contralateral kidneys in the staining of renal tubules: (a) the intensity of fluorescent staining in cortical and medullary collecting tubules of the hydronephrotic kidney was increased and (b) cyclooxygenase antigenicity appeared in the thin limbs of Henle's loop in the hydronephrotic organ. Although alterations in prostaglandin formation by the renal vasculature have been documented previously, our results indicate that ureteral obstruction also causes increased prostaglandin synthesis by renal tubules.     

Increased renal tubular synthesis of prostaglandins in the rabbit kidney in response to ureteral obstruction.

The hydronephrotic rabbit kidney exhibits elevated basal prostaglandin synthesis and supersensitivity to peptide stimulation of vascular prostaglandin and thromboxane formation. In this study the distribution of the prostaglandin-forming cyclooxygenase in hydronephrotic and contralateral rabbit kidneys following one and four day ureteral obstructions was compared using immunohistofluorescence. No alterasions were detected in the distribution or intensity of cyclooxygenase-positive fluorescence in the renal vasculature in response to ureteral obstructions. However, two significant differences were noted between hydronephrotic and contralateral kidneys in the staining of renal tubules: (a) the intensity of fluorescent staining in cortical and medullary collecting tubules of the hydronephrotic kidney was increased and (b) cyclooxygenase antigenicity appeared in the thin limbs of Henle's loop in the hydronephrotic organ. Although alterations in prostaglandin formation by the renal vasculature have been documented previously, our results indicate that ureteral obstruction also causes increased prostaglandin synthesis by renal tubules.     

Effect of chloride and diamide on serum angiotensin i-converting enzyme activity from eight mammalian species

1. The effect of chloride on serum angiotensin I-converting enzyme (ACE) activity was characterized in eight mammalian species: dog, guinea pig, hamster, human, mouse, rabbit, rat, and sheep.2. Optimum chloride concentrations varied from 300 mM for rabbit to 1700 mM for hamster.3. The increments with these optimum concentrations with respect to 100 mM chloride concentration were from 1.4-fold in rabbit to 7.9-fold in hamster and dog.4. There was no correlation between serum chloride concentration or serum ACE activity and optimum chloride concentration.5. Serum ACE increased only in humans with diamide pretreatment suggesting the presence of endogenous inhibitors.     

Species heterogeneity of hepatic alpha 1-adrenoceptors: alpha 1A-, alpha 1B- and alpha 1C-subtypes.

alpha 1-Adrenergic activation stimulated phosphorylase and phosphoinositide turnover in hepatocytes from guinea pigs, rats and rabbits. Chlorethylclonidine inhibited these effects in rat and rabbit cells but not in guinea pig hepatocytes; low concentrations of 5-methyl urapidil blocked the alpha 1 actions in guinea pig and rabbit liver cells, but not in rat hepatocytes. Binding competition experiments also showed high affinity for 5-methyl urapidil in liver membranes from guinea pigs and rabbits and low affinity in those from rats. The data indicated that guinea pig hepatocytes express alpha 1A-, rat hepatocytes alpha 1B- and rabbit hepatocytes alpha 1C- adrenoceptors. This was confirmed by Northern analysis using receptor subtype-selective probes.     

Guinea pig complement potently measures vibriocidal activity of human antibodies in response to cholera vaccines

The vibriocidal assay using guinea pig complement is widely used for the evaluation of immune responses to cholera vaccines in human clinical trials. However, it is unclear why guinea pig complement has been used over human complement in the measurement of vibriocidal activity of human sera and there have not been comparison studies for the use of guinea pig complement over those from other species. Therefore, we comparatively investigated the effects of complements derived from human, guinea pig, rabbit, and sheep on vibriocidal activity. Complements from guinea pig, rabbit, and human showed concentration-dependent vibriocidal activity in the presence of quality control serum antibodies. Of these complements, guinea pig complement was the most sensitive and effective over a wide concentration range. When the vibriocidal activity of complements was measured in the absence of serum antibodies, human, sheep, and guinea pig complements showed vibriocidal activity up to 40-fold, 20-fold, and 1-fold dilution, respectively. For human pre- and post-vaccination sera, the most potent vibriocidal activity was observed when guinea pig complement was used. In addition, the highest fold-increases between pre- and post- vaccinated sera were obtained with guinea pig complement. Furthermore, human complement contained a higher amount of V. cholerae- and its lipopolysaccharide-specific antibodies than guinea pig complement. Collectively, these results suggest that guinea pig complements are suitable for vibriocidal assays due to their high sensitivity and effectiveness to human sera.     

Cytotoxic effects of normal sera on lymphoid cells. I. Antibody-independent killing of heterologous thymocytes by guinea pig, rabbit, and human sera: role of the alternative pathway of complement activation.

The mechanisms whereby normal sera may cause the death of xenogeneic lymphoid cells in vitro have been reviewed in this study using guinea pig, rabbit and human sera as the source of activity and rat and mouse thymocytes as target cells. In all of the combinations analyzed the cytotoxic reactions were found to be mediated by complement (C) as evidenced by sensitivity of the sera towards either heat inactivation (56 °C, 30 min) or treatment with cobra venom factor or sodium ethylenediaminotetraacetate (EDTA). C activity was provided via the alternative pathway in every instance: (i) both C4-deficient guinea pig serum and C2-deficient human serum displayed cytotoxicity on the target cells; (ii) sera from all three sources were active in the absence of free Ca²⁺, which is required to activate C via the classical pathway; and (iii) GPS incubated at 50 °C for 20 min to destroy the activity of factor B of the alternative pathway lacked significant cytotoxic activity while still able to lyse sensitized sheep red blood cells, a reaction proceeding via the C142 pathway. Two independent lines of evidence appeared to exclude the possible role of antibodies in nonspecific serum cytotoxicity. First, the cytotoxic capacities and the titers of guinea pig and rabbit sera were not significantly affected after absorption with target cells in the presence of EDTA, i.e., in the absence of free divalent cations, a condition which does not interfere with antigen antibody binding. By contrast, the activity was eliminated when absorption was performed in the absence of chelating agents or in the presence of a selective Ca²⁺ chelator, sodium ethyleneglycoltetraacetate, plus excess Mg²⁺ These observations also highlight the Mg²⁺-dependence of the removal of activity by absorption. Second, γ-globulins isolated from a highly cytotoxic guinea pig serum were not toxic for rat thymocytes when tested in the presence of rat C. These results suggest that conventional antibodies, whether of “natural” origin or otherwise, are unlikely to play a role in serum-produced nonspecific cytotoxicity. Furthermore, and since incubation of human serum with rat or mouse thymocytes produced conversion of factor B, “absorption” of cytotoxic activity would seem to be more likely a consequence of the consumption of C activity via the C3 shunt than of the removal of any antibodies.     

Does diaminobenzidine demonstrate prostaglandin synthetase?

Summary A method histochemical localization of prostaglandin synthetase using DAB, potassium cyanide and polyunsaturated fatty acid has been revised. The arachidonic acid-induced DAB oxidation observed in the secretory epithelium of sheep vesicular glands and in collecting tubules as well as interstitial cells of rabbit kidney medulla was found to be insensitive to antiinflammatory cyclooxygenase (formerly referred as prostaglandin synthetase) inhibitors, such as indomethacin, aspirin, mefenamic acid and paracetamol, whereas aminotriazole caused complete inhibition of the reaction. Furthermore, DAB was oxidized in the presence of polyunsaturated fatty acids inconvertible to prostaglandins (linoleic and linolenic acid) as well as in the presence of H₂O₂ — in the latter case reaction possessed identical features with that induced by fatty acids. Ultrastructurally, the reaction product was localized on the membranes of nuclear envelope and endoplasmic reticulum. On the ground of the results obtained a hypothesis is presented, that the polyunsaturated fatty acid-induced DAB oxidation is due to a peroxidatic activity of the investigated tissues. Possible relations between such peroxidatic activity and prostaglandin biosynthesis are discussed.     

Protein binding of cortisol by means of competitive adsorption: application to cortisol binding by serum of sixteen eutherian mammals.

1. Binding of 3H-cortisol by serum proteins by means of competitive adsorption was relatively high by serum of the gerbil, human, rabbit, sheep, tree shrew, hamster, rhesus monkey and horse. 2. A somewhat lower binding was observed by serum proteins of the baboon, cattle, dog, rat and cat. 3. Serum taken from either the mouse, guinea pig or pig gave very flat binding curves, specific binding not exceeding 5% of added 3H-cortisol. 4. It is concluded that the measurement of protein-binding of 3H-cortisol by means of competitive adsorption is a reliable method for serum of most eutherian species but is unsuited if serum of the mouse, guinea pig or pig is used.     

Binding of [3H]Ro 5–4864 and [3H]PK 11195 to Cerebral Cortex and Peripheral Tissues of Various Species: Species Differences and Heterogeneity in Peripheral Benzodiazepine Binding Sites

The binding of [3H]PK 11195 and [3H]Ro 5-4864 to membrane preparations from cerebral cortex and peripheral tissues of various species was studied. [3H]PK 11195 (0.05-10 nM) bound with high affinity to rat and calf cerebral cortical and kidney membranes. [3H]Ro 5-4864 (0.05-30 nM) also successfully labeled rat cerebral cortical and kidney membranes, but in calf cerebral cortical and kidney membranes, its binding capacity was only 3 and 4%, respectively, of that of [3H]PK 11195. Displacement studies showed that unlabeled Ro 5-4864, diazepam, and flunitrazepam were much more potent in displacing [3H]PK 11195 from rat cerebral cortex and kidney membranes than from calf tissues. The potency of unlabeled Ro 5-4864 in displacing [3H]PK 11195 from the cerebral cortex of various other species was also tested, and the rank order of potency was rat = guinea pig greater than cat = dog greater than rabbit greater than calf. Analysis of these displacement curves revealed that Ro 5-4864 bound to two populations of binding sites from rat and calf kidney and from rat, guinea pig, rabbit, and calf cerebral cortex but to a single population of binding sites from cat and dog cerebral cortex. Using [3H]PK 11195 as a ligand, the rank order of binding capacity in cerebral cortex of various species was cat greater than calf greater than guinea pig greater than rabbit greater than dog greater than rat, whereas when [3H]Ro 5-4864 was used, the rank order of binding capacity was cat greater than guinea pig greater than rat greater than rabbit greater than calf greater than dog.