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The objectives of this study were to verify the presence of membranous vesicles (MV) in canine seminal plasma by mean of transmission electron microscopy (TEM), to describe the ultrastructural characteristics and to identify some enzymatic activity associated with them. Semen samples, collected by digital manipulation from dogs with proven fertility, were pooled and used for membrane vesicles preparation according to conventional procedures. TEM observations showed the existence of vesicular membranous structures of more or less spherical shape with different sizes. These vesicles were surrounded by a single-, double- or multiple-layered laminar membranes. The mean vesicle diameter was 117.6 ± 86.9 nm ranging from 24.4 to 716.6 nm. Enzyme activity determinations showed the presence of adenosine deaminase, 5'-nucleotidase, ADPase, ATPase, dipeptilpeptidase IV, alkaline phosphatase, total acid phosphatase and prostatic acid phosphatase, while the aminopeptidase activity was absent. In conclusion, results of this study, compatible with results from other mammals, showed for the first time the presence of MV, their ultrastructural and enzymatic characteristics in dog seminal plasma.

Biochemical studies demonstrate that the NO-releasing-aspirin derivative (NCX4016) stimulates soluble guanylate cyclase (sGC) activity and increases cyclic GMP (cGMP) in human platelet and monocytes by releasing NO. In the present study, an ultracytochemical technique for electron microscopy was used to investigate the effects of NCX4016 (2 mM) on sGC activity in rat thoracic aorta, using sodium nitroprusside (0.01 mM) as reference NO-donor. Guanylyl-imidodiphosphate sodium salt [Gpp(NH)p], a synthetic non-hydrolyzable analogue of GTP, was used as sGC substrate. NO-activated sGC released imidodiphosphate ions which were precipitated with lead ions, giving rise to deposits of electron-dense granules (reaction product). Ultracytochemistry allowed us to demonstrate that NCX4016 stimulated sGC activity in smooth muscle cells, and particularly in vascular endothelial cells, as sodium nitroprusside did. This result could explain the protective effects of chronic treatment with NCX4016 on aortic endothelium of diabetic rats demonstrated by scanning and transmission electron microscopy.

The effect of a nitric oxide-donating aspirin derivative, 2-acetoxy-benzoate 3-(nitroxy-methyl)phenyl ester (NCX 4016), and aspirin on the aortic endothelium of diabetic rats was investigated by using scanning and transmission electron microscopy. Control and streptozotocin-treated rats were used. Metabolic control was assessed by measuring blood and urine metabolites, and 24-h urine volume. The ultrastructural study was performed after 7 weeks of diabetes and 6 weeks of therapy. Streptozotocin treatment induced a persistent hyperglycemia which was not influenced by the pharmacological treatments. Values of blood metabolites were in line with the diabetic status. Both scanning and transmission electron microscopy revealed that aortic endothelium was severely damaged in all diabetic rats except for the NCX 4016 treated ones. Our data document the protective effects of NCX 4016 on the vascular endothelium of diabetic rats. Since aspirin had no protective action, NCX 4016 may have exerted its beneficial action by releasing nitric oxide.

The ultracytochemical localization of adenylate cyclase (AC) and guanylate cyclase B (GC-B) and C (GC-C) activity was studied after stimulation with pituitary adenylate cyclase activating peptide, C-type natriuretic peptide and guanylin, respectively, in normal human respiratory nasal mucosa and mucosa of nasal polyps. To demonstrate these enzymatic activities, we employed enzyme-ultracytochemical methods for electron microscopy. Both normal and pathologic nasal mucosa contained AC, GC-B and GC-C activity. In the upper portion of respiratory epithelium, the enzymes were detected on ciliary and microvillar membranes. In ciliary membranes, GC-B was the predominant form expressed. In goblet cells and in glands of the lamina propria, enzymatic activities were localized mainly on plasma membranes and on membranes lining secretory granules. The results did not reveal any evident differences between the enzymatic activities in normal and pathological nasal mucosa and suggest complementary activities for these enzymes and their stimulators in the regulation of mucociliary transport and glandular secretion.

The ultracytochemical localization of membrane-bound guanylate cyclases A and C, stimulated by atrial natriuretic peptide and guanylin respectively, has been studied in human sweat glands. The results showed that the peptides stimulated guanylate cyclases A and C in both eccrine and apocrine glands. In the secretory cells, enzymatic activity was present on the plasma membranes and on intracellular membranes involved in the secretory mechanism. In eccrine glands, the cells of the excretory duct also presented enzymatic activity on the plasma membranes. In both glands, myoepithelial cells, surrounding the secretory cells, exhibited only guanylate cyclase A activity. These localizations of enzymatic activity suggest a role for both atrial natriuretic peptide and guanylin in regulating glandular secretion.

Intestinal guanylate cyclase C is activated by guanylin, an endogenous peptide. This activity seems to be modulated by adenine nucleotides, the ions Mg2+ and Mn2+, and pH. In this study, we report an ultracytochemical method for the localization of guanylate cyclase C activity at the electron microscope level. We studied the enzymatic activity in the presence or absence of guanylin and/or ATP, in the presence of the ions Mg2+ or Mn2+, and at different pH levels. The greatest distribution of enzymatic activity was detected in samples incubated at pH 8 and 7.4 in the presence of guanylin, Mg2+ and ATP. Guanylate cyclase C activity was detected at the surface epithelium of stomach and intestine, and in liver, exocrine pancreas and parotid gland. In the intestine, enzymatic activity was more widely distributed in the duodenum than in the jejunum-ileum and colon. In the small intestine, activity was more evident in the upper portion than in the basal portion of the villus. In samples incubated at pH 8 and 7.4 in the absence of ATP, enzymatic activity was detected only in small intestine, liver and exocrine pancreas. Enzymatic activity was present in duodenum incubated at pH 8 and 7.4 in the presence of Mn2+ and in the presence or absence of ATP. No samples incubated in all these experimental conditions but at pH 5 or samples incubated in the presence of guanylin only or in the absence of guanylin, displayed guanylate cyclase C activity. Our results suggest that a complete ultracytochemical detection of guanylate cyclase C activity requires guanylin as stimulator, and incubation in the presence of Mg2+ and ATP at pH 8 and 7.4.

The ultracytochemical localization of guanylate cyclases A and B activity has been studied after stimulation with atrial natriuretic peptide and C-type natriuretic peptide in light- and dark-adapted retinas and pigmented epithelium. The results showed that both peptides stimulated guanylate cyclases A and B activity in light-adapted retinas only. Guanylate cyclases A and B activity was detected on plasma membrane of body of photoreceptors, bipolar, horizontal and ganglion cells, on plasma membranes of interneuronal connections at plexiform layers and on the plasma membrane of fibres at the nerve fibres layer. Independently of the light-or dark-adapted state, the pigmented epithelium also presented guanylate cyclases A and B activity on basal and lateral plasma membranes.

The ultracytochemical localization of membrane-bound guanylate cyclases A and B has been studied after stimulation with atrial natriuretic peptide, C-type natriuretic peptide and brain natriuretic peptide in the gastrointestinal tract of rat. The two isoforms are stimulated differently by the three peptides. The results showed that the atrial and C-type natriuretic peptides stimulated guanylate cyclase activity, whereas the brain peptide seemed not to activate enough of the enzyme to detect. The guanylate cyclase activity had a wider distribution in stomach and small intestine than in large intestine; nevertheless, the reaction product of guanylate cyclase A activity had a wider localization in the stomach, whereas the reaction product of guanylate cyclase B activity had a wider distribution in the small intestine. In the small and large intestine, we detected mostly similar localizations of guanylate cyclase activity irrespective of the peptide used; in the stomach the reaction products of guanylate cyclase A and B were detected in different cell types or in different sites of the same cell. In all the gastrointestinal tract, guanylate cyclase activity was detected mainly in three types of cells: exocrine and endocrine cells; undifferentiated and mature epithelial cells; and smooth muscle cells. These localizations of guanylate cyclase activity suggest its role in regulating glandular secretion, cellular proliferation and muscular activity.

A minireview is presented on the ultracytochemical localization of membrane-bound guanylate cyclase (GC) in various tissues and in cultured cells after activation with three natriuretic peptides, the atrial natriuretic factor (ANF), the brain natriuretic peptide (BNP), and the C-type natriuretic peptide (CNP). GC, two subtypes of which have been recently identified, is the receptor for these peptides. The GC isoforms are differently stimulated by ANF, BNP and CNP. Under our experimental conditions, the natriuretic peptides were strong activators of GC since samples incubated without natriuretic peptides do not reveal any cyclase activity. The natriuretic peptide-stimulated GC activity was studied in rat kidney, lung, adrenal gland and neurohypophysis, in rabbit platelets, in lamb olfactory mucosa, and in rat C6 glioma cells. On the basis of the subcellular GC localization some additional functions of peptides are hypothesized.

The ultracytochemical localization of particulate guanylate cyclase has been studied in lamb olfactory mucosa after activation with rat atrial natriuretic factor (rANF), porcine brain natriuretic peptide (pBNP), porcine C-type natriuretic peptide (pCNP) or rat brain natriuretic peptide (rBNP). Particulate guanylate cyclase is the receptor for these peptides and recently two subtypes of the cyclase have been identified. These isoforms are stimulated differently by ANF, BNP and CNP. Under our experimental conditions, rANF, pCNP and pBNP were strong activators of particulate guanylate cyclase in lamb olfactory mucosa, as demonstrated by the presence of reaction product. Samples incubated in basal conditions without rANF, pCNP or pBNP, or samples incubated in presence of rBNP did not reveal any cyclase activity. The rANF-stimulated cyclase activity was localized in the apical portion of olfactory epithelium. pCNP-stimulated guanylate cyclase was detected to the lamina propria in association with secretory cells of Bowman's glands and with cells in close relation with Bowman's glands (elongated cells and myoepithelial cells). The cyclase activity stimulated by pBNP was limited to cells of Bowman's glands. The present data indicate that ANF and CNP are recognized by different receptors and that BNP and CNP bind to the same receptor.

The cellular and subcellular localization of annexins V and VI, two members of a superfamily of Ca(2+)-dependent phospholipid- and membrane-binding proteins, was investigated in chorionic villi of human placentae of different gestational ages by postembedding immunocytochemistry at the electron microscope level. All cell types of placental villi, i.e., the syncytiotrophoblast, Langhans cells, Hofbauer cells, fibroblasts, and capillary endothelial cells, appeared to express the two proteins, irrespective of the gestational age. By immunogold particle counts, annexin V was observed to be 2-3 times as much abundant as annexin VI. Syncytiotrophoblast cells appeared to contain the largest amounts and Langhans cells appeared to contain the least amounts of annexins V and VI, as judged by immunocytochemistry. The two proteins were found associated with plasma, Golgi, and vacuolar membranes, and with membranes of the endoplasmic reticulum, as well as diffusely in the cytoplasm. Annexin V appeared to be distributed in nearly equal proportions between cell membranes and the cytoplasm in stromal cells and to be about 30% associated with cell membranes in trophoblast cells, whereas annexin VI appeared almost equally distributed between cell membranes and the cytoplasm in trophoblast and stromal cells. Also, annexins V and VI appeared to be more abundant in trophoblast cells than in stromal cells. The present data strongly support the idea that placenta is a preferential site of annexin-regulated activities, and suggest that annexins V and VI are actively involved in the Ca(2+)-dependent regulation of membrane processes in trophoblast cells.

We studied the cytochemical localization of particulate guanylate cyclase (GC) activity after stimulation with atrial natriuretic factor (ANF) in rat lung, at the electron microscope level. Samples incubated in the absence of ANF did not reveal any GC reaction product. These results indicate that ANF is a strong activator of the enzyme in this organ. In intrapulmonary bronchi, the ANF-activated GC reaction product was localized on mucus secreting goblet cells. GC was seen in bronchioles, alveoli and capillaries. All of the GC reaction product was associated with plasma membranes of Clara cells, of great alveolar cells and of endothelial cells in alveolar capillaries. Our data suggest that, by activation of particulate GC, ANF acts directly on cells where Na+ reabsorption occurs.

Previous biochemical and cytochemical studies have indicated that in human term placenta the enzyme guanylate cyclase (GC) is associated mostly with the cytosolic fraction of homogenates and localized on the syncytiotrophoblast microvillous border. In the present study we have shown cytochemically the GC particulate form in early human placenta using guanylyl-imidodiphosphate [Gpp(NH)p] as substrate and NaN3 as activator. In samples of placental villi taken from the 6th to 12th week of pregnancy, the GC reaction product was always found on the apposing Langhans cytotrophoblast and syncytiotrophoblast plasma membranes. Furthermore, GC was present on cells in mitosis of the Langhans cytotrophoblast. From the 11th week GC was also visible on basal plasma membranes of Langhans cytotrophoblast and on endothelial cells of fetal capillaries. In samples of human term placenta GC was detectable on the syncytiotrophoblast microvillous border. This suggests a shift of enzyme localization during pregnancy.