Modulation of granulocyte responses in three-spined sticklebacks Gasterosteus aculeatus infected with the tapeworm Schistocephalus solidus

Leukocytes isolated from the head kidney and peripheral blood of 3-spined sticklebacks Gasterosteus aculeatus L. were analysed by means of flow cytometry during infection with the tapeworm Schistocephalus solidus (Müller, 1776). Although parasites increased their body weight continuously throughout the observation period (98 d), proportions of granulocytes increased in blood and head kidney only up to Day 63 post-infection (p.i.). Thereafter, declining proportions of granulocytes were observed in both organs. Thus the relative decrease in granulocyte number was not correlated to a decline in the parasitic load of the fish. To investigate a possible modulatory impact of S. solidus on granulocyte function, head kidney leukocytes were isolated at times before Day 63 p.i. and tested in vitro for their capacity to produce reactive oxygen species (ROS). Head kidney leukocytes from S. solidus-infected fish, analysed immediately after isolation (ex vivo, Day 40 p.i.), exhibited a higher ROS production when stimulated with phorbol myristate acetate (PMA), than leukocytes from naive, sham-treated control fish and fish that had resisted or cleared the infection (exposed but not infected). The latter showed an increased spontaneous ROS production that was not correlated to the numbers of granulocytes present in the head kidney isolates. In infected sticklebacks, spontaneous and PMA-induced ROS production was significantly correlated with numbers of granulocytes present in the head kidney isolates, suggesting that elevated ROS production was due to higher numbers of responding cells rather than an increased capacity of single cells. In vitro, after cultivation for 4 d in the presence of pokeweed mitogen (PWM) or extracts from S. solidus, head kidney leukocytes from control fish showed an increased ROS production and phagocytic activity compared with non-stimulated control cultures. In contrast, head kidney leukocytes from infected fish isolated on Days 48 and 44 p.i., failed to respond to S. solidus antigens in vitro. During S. solidus infection, granulocyte mobilisation resulted in elevated numbers of these cells in head kidneys, but the lack of an in vitro response to S. solidus antigens indicates an in vivo priming of granulocytes by the parasite. These observations may reflect the ability of S. solidus to impair the host's immune response once the parasite is developing in the body cavity of G. aculeatus.     

Phagocytosis by carp granulocytes; in vivo and in vitro observations

The phagocytic ability of carp (Cyprinus carpio L.) granulocytes was evaluated in vivo and in vitro. In suspensions of head kidney cells, neutrophil granulocytes incorporated both latex beads and coccidian merozoites. In intestinal tissues from carp with a Goussia carpelli infection, all granulocyte cell types (neutrophils and cells of the basophilic-eosinophilic complex) phagocytosed cell detritus and coccidian developmental stages, mainly merozoites.     

Adaptability of carp neutrophilic granulocytes to environmental temperatures: spontaneous cytotoxic activity and cell-adherent activity

This study observed the adaptability of carp neutrophilic granulocytes possessing spontaneous cytotoxic activity to different environmental temperatures. To study the adaptability of neutrophilic granulocytes, two different temperatures (25° C and 10° C) were selected, both for rearing and forin vitroassays, in which the cytotoxicity and the adherent rate against K562 target cells were measured. The cytotoxicity and adherent rate of neutrophilic granulocytes from carp kept at 25° C for 30 days were higher when assayed at 25° C than when assayed at 10° C. On the other hand, in carp acclimated from 25° C to 10° C, the cytotoxicities and adherent rates, when assayed at 25° C, decreased with increasing acclimation times, eventually becoming smaller than the values obtained when assayed at 10° C. After the fish kept at 10° C for a long period were re-acclimated to 25° C, these activities assayed at 25° C again became higher than the activities assayed at 10° C. These results indicated that carp neutrophilic granulocytes adapt their cytotoxic activity and adherent activity to different environmental temperatures. A change in cellular composition in the head kidney was also observed in carp kept at different environmental temperatures. The percentage of neutrophilic granulocytes became higher and lymphocytes became lower in carp that were kept at 10° C for a long period compared with carp that were kept at 25° C for a long period.     

Identification of the site of glucocorticoid action on neutral amino acid transport in superficial nephrons of rat kidney

Summary. Glucocorticoid hormones enhance the reabsorptive capacity of filtered amino acids in rat kidney, as it was shown in previous in vivo clearance experiments. In the present study, the site of glucocorticoid action on neutral amino acid transport in superficial nephrons of rat kidney was investigated using in vivo micropuncture technique. Adult female Wistar rats were treated with dexamethasone (DEX), and fractional excretion of L-glutamine (L-Gln) and L-leucine (L-Leu) were determined and related to inulin after microinfusion into different nephron segments. DEX reduced fractional excretion of both neutral amino acids as a sign of enhanced reabsorptive capacity. The site of main DEX action on L-Leu reabsorption has been localized in the proximal straight tubule. However, in the case of L-Gln, the inhibition of γ-glutamyltranspeptidase (γ-GT) by administration of acivicin indicated the importance of this brush border enzyme in reduced L-Gln excretion. DEX enhanced γ-GT activity by tubular acidification. It can be presumed a DEX-inducible transport system for neutral amino acids mainly localized in proximal straight tubules of rat kidney. Received July 8, 1999     

Gut-associated lymphoid tissue (GALT) of the goldfish,Carassius auratus

The head kidney and spleen are major sites of haemopoiesis in fish; a secondary center is found in loose connective tissue of the intestine. In this study we determined the nature of gut-associated haemopoietic tissue in the goldfish, Carassius auratus, using light and electron microscopy. This tissue is a loose stroma of reticular cells and fibers vascularized by capillaries, venules, and arterioles. The cellular population includes lymphoblasts, small and medium-sized lymphocytes, plasmocytes, macrophages, and various granulocytes. The most abundant granulocyte is the mast cell, whose large granules stain with Alcian blue and toluidine blue. Heterophils are found in the intestinal connective tissue as well as two other granulocytes: one with ovoid granules having dense parallel lamellae and another with granules containing crystalline inclusions. Immature forms of both granulocytes were also noted. Macrophages containing phagocytosed debris were often located close to the epithelium; they were observed forming clusters with lymphocytes. The epithelium contained a number of migrating leucocytes including lymphocytes and lymphoblasts, macrophages, and heterophils. Although many granulocytes were found in the connective tissue, granulopoiesis does not seem to be a major function. Gut-associated haemopoietic tissue in goldfish resembles diffuse lymphoid tissue and may be involved in intestinal immune responses.     

Breeding-related seasonal changes in immunocompetence, health state and condition of the cyprinid fish, Rutilus rutilus, L.

Seasonal changes in immunocompetence are predicted by the hypothesized trade-off between reproduction and self-maintenance, whereby immune function is a measure of self-maintenance and reproductive effort is seasonally dependent. We examined seasonal patterns in immunological, haematological and body condition parameters for male and female freshwater fish. In two different populations, the relative size of the spleen and the chemotaxic migration activity of head kidney granulocytes decreased immediately before and after spawning, respectively. Those decreases were accompanied by an increase in haematocrit values shortly before and after spawning in both populations and a decrease in the relative body weight in one population, possibly due to physiological stress caused by increased activity during the spawning period. Breeding-related changes in other measures studied (phagocytosis activity of head kidney granulocytes, blood IgM concentration, white cell count and red cell count) were absent or inconsistent. Some of the results are in line with the idea of a trade-off between reproduction and immune defence. The present data suggest that the differential responses of the immune system may have different temporal patterns, which should be taken into account in immunoecological studies.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 78 , 117−127.     

cDNA sequence and tissue expression of an antimicrobial peptide, dicentracin; a new component of the moronecidin family isolated from head kidney leukocytes of sea bass, Dicentrarchus labrax

A 483-bp cDNA was isolated from sea bass (Dicentrarchus labrax) head kidney leukocytes, dicentracin, using PCR primers designed from conserved moronecidin domains. Gene bank analysis revealed that dicentracin cDNA belongs to the moronecidin family. As deduced from alignment with Morone chrysops moronecidin, the precursor of 79 aa appeared to be composed of a signal peptide of 22 aa, followed by the mature AMP (antimicrobial peptide) of 22 aa named dicentracin, and a C-terminal extension of 35 aa. Dicentracin precursor displayed 3 aa substitutions with other moronecidin sequence but none in the mature peptide sequence. Using in situ hybridization assay, dicentracin gene expression was observed in 68–71% of peripheral blood leukocytes, kidney leukocytes or peritoneal cavity leukocytes without significant statistical differences. Dicentracin mRNA was observed in most of the granulocytes, as well as in monocytes from both peripheral blood and head kidney, and in macrophages from peritoneal cavity. No expression was observed in thrombocytes or in lymphocytes.     

In vivo distribution of cryogenically preserved guinea pig granulocytes

Granulocytes were isolated from whole blood of guinea pigs by counterflow centrifugation and labeled with [¹⁴C]diisopropylfluorophosphate ([¹⁴C]DFP). One-half of the labeled cells was injected intravenously via the femoral vein into a guinea pig, while the other half was cryogenically preserved with 5% dimethyl sulfoxide (DMSO), 6% hydroxyethyl starch (HES), and 4% human albumin, at a rate of 4 °C per minute by storage at ?80 °C and then stored for 3 days at ?80 °C. Ninety percent of the isolated granulocytes were recovered after cryogenic preservation, thawing, and washing. Aliquots before injection all produced fluorescein from fluorescein diacetate and excluded ethidium bromide. Latex ingestion was 78% and yeast ingestion was 75%. The frozen-thawed-washed-resuspended labeled granulocytes were injected into a second guinea pig. Paired animals sacrificed 35 min after injection were examined in whole-body sections for distribution of radiolabeled granulocytes to the tissues. In two pairs of animals, activity was found in the lung, liver, spleen, and kidney. The technique does not permit a distinction between fresh and cryopreserved granulocytes although there was a greater deposition of fresh cells in the liver and spleen. No activity was found in the blood of the vena cava in animals with either fresh or frozen cells. An animal injected with free [¹⁴C]DFP revealed a vascular distribution with high activity in blood, lung, and kidney, and less activity in the liver and spleen. The data indicate that radiolabeled, cryogenically preserved guinea pig granulocytes exhibited a tissue distribution qualitatively similar to fresh granulocytes, and free [¹⁴C]DFP infused without granulocytes differed qualitatively and quantitatively from fresh and cryopreserved granulocytes.     

Oxidation of salicylates by stimulated granulocytes: evidence that these drugs act as free radical scavengers in biological systems

Although salicylates have been used for centuries as treatment of inflammatory diseases, the mechanism of action of these drugs is still not clear. Aspirin (acetylsalicylic acid) and other nonsteroidal anti-inflammatory drugs (NSAID) inhibit prostaglandin biosynthesis, a property that appears to explain part of their anti-inflammatory activity. However, this mechanism does not appear to explain the anti-inflammatory properties of salicylic acid, which is a major metabolite of ASA in vivo. Results of prior studies in our laboratory have established that benzoic acid, the parent compound of the salicylate group of drugs, is decarboxylated and hydroxylated by the hydroxyl free radical (OH.) produced by stimulated granulocytes. These observations suggested that salicylates might be similarly metabolized by granulocytes. If so, the capacity of salicylates to rapidly react with OH. might relate directly to their known anti-inflammatory properties. Preliminary experiments established that salicylic acid and aspirin were decarboxylated by the hydroxyl free radical generated by the enzyme system xanthine-xanthine oxidase. We then studied the metabolism of salicylates by human granulocytes. Unstimulated granulocyte suspensions did not oxidize ASA or salicylic acid. However, suspensions stimulated by opsonized zymosan particles rapidly oxidized both substrates in pharmacological concentrations. The rate of oxidation of salicylic acid was 16-fold higher than benzoic acid, whereas the rate of oxidation of ASA was four-fold higher. The reaction was oxygen dependent and could be inhibited by known hydroxyl scavengers, particularly dimethylthiourea. The reaction could also be inhibited by superoxide dismutase and azide, indicating that O-2 and heme or an iron-dependent enzyme were required for the reaction. The reaction was not impaired by compounds known to react with the HOCL and the chloramines generated by stimulated PMN. Furthermore, salicylic acid in high concentrations did not impair the HMPS pathway, the production of O-2 or the production of H2O2 by granulocytes. These data provide evidence that salicylates are rapidly oxidized by the hydroxyl free radical produced by granulocytes and not O-2, H2O2, or HOCL. This capacity of salicylates to react rapidly and selectively react with OH. may directly relate to their anti-inflammatory properties. In addition, results of our experiments indicate that stimulated granulocytes acquire the capacity to metabolize these drugs. Therefore, several metabolites of salicylates may be produced at a site of inflammation, all of which may have altered biological activity compared with the parent compound.     

Cytotoxicity of white blood cells activated by granulocyte-colony-stimulating factor,granulocyte/macrophage-colony-stimulating factor and macrophage-colony-stimulating factor against tumor cells in the presence of various monoclonal antibodies

Unconjugated monoclonal antibodies (mAb) kill tumor cells in vivo by activating immune functions. One of these is ADCC (antibody-dependent cellular cytotoxicity). The efficacy of mAbs might be augmented if the cytotoxic capacity of the effector cells could be increased. In this study the augmenting effect of granulocyte-colony-stimulating factor (G-CSF), granulocyte/macrophage(GM)-CSF and macrophage(M)-CSF was analyzed. Effector cells [peripheral blood mononuclear cells (PBMC) or granulocytes] were activated for 4–6 h by the respective CSF and assayed in an 18-h Cr⁵¹-release assay. Human colorectal, lymphoma, glioma and melanoma cell lines were target cells. Mouse mAbs of different isotypes, as well as chimeric and humanized mAbs, were used. mAbs having the human Fc part of the IgG molecule were the most effective. The killing capacity of PBMC as well as of granulocytes was statistically significantly enhanced when mAbs were added. M-CSF and GM-CSF were the best CSF for augmenting the lytic capacity of PBMC in ADCC. G-CSF had no significant effect on PBMC. Spontaneous cytolysis of PBMC was significantly augmented only by M-CSF. Granulocytes were, in general, significantly less effective than PBMC but may be equally effective killer cells together with mouse or human mAbs of the IgG1 isotype, particularly against melanoma cells. Granulocytes may also be significantly stimulated to increased lytic capacity when activated with G-CSF or GM-CSF. On the basis of the present evaluation, clinical trials in tumor patients are warranted, combining mAbs with GM-CSF or M-CSF. Preference might be given to GM-CSF as this cytokine activates both PBMC and granulocytes.     

Compartmentation of hexokinase in human blood cells characterization of soluble and particulate enzymes

The isozyme distribution, kinetic properties and intracellular localization of hexokinase (ADP: D-hexose-6-phosphotransferase, EC 2.7.1.1) were studied in erythrocytes, blood platelets, lymphocytes and granulocytes. Soluble and particulate fractions were separated by a rapid density centrifugation method after controlled digitonin-induced cell lysis. In lymphocytes and platelets the major part of total activity was particle-bound (78 and 88%, respectively). In granulocytes and erythrocytes most of the hexokinase activity was found in the cytosol. All cell types, except granulocytes, contain mainly the type I isozyme. Platelets contain only type I hexokinase, while in lyphocytes a minor amount of type III is present in the soluble fraction (less than 10% of total activity). The major constituent of granulocytes is type III hexokinase (70–80% of total activity), the remaining 20–30% is type I hexokinase. Erythrocytes contain a multibanded type I hexokinase. The substrate affinities of the type I hexokinase do not differ significantly between the different cell types or between soluble, bound and solubilized fractions. Only soluble hexokinase from lymphocytes shows a slightly decreased K_m apparent for glucose (P < 0.05).     

Enzyme characterisation of the circulating haemocytes of the carpet shell clam,Ruditapes decussatus(Mollusca:bivalvia)

The occurrence of a number of lysosomal enzymes (Proteases, glycosidases, phosphatases, and esterases) inRuditapes decussatushaemocytes was demonstrated by cytochemical and colorimetric techniques. The levels of 18 enzymes tested monthly varied through the study period (18 months), although they did not conform to a seasonal pattern of variation. No important effect of clam age on enzyme activity levels of haemocytes was detected. In those cytochemical assays in which distinction between granulocytes and hyalinocytes was possible, lysosomal enzymes were only found in granulocytes. Phosphatase was detected inside cytoplasmic granules of granulocytes, suggesting the granules to be lysosomes. NADPH oxidase was not detected in clam haemocytes, which is consistent with the absence of oxidative metabolism coupled with phagocytosis in haemocytes of this clam species. Levels of lysozyme detected inside haemocytes were higher than in serum.     

Stimulating effect ofStreptococcus faecalis on phagocytosis in gnotobiotic piglets

The concentration of active phagocytes in peripheral blood remained in germfree pigs up to the age of one year approximately at the same level as found at the age of 7 d and did not exceed 0.3×10⁹/L of blood, whereas a steady increase was established in conventional pigs. Monoassociation of gnotobiotic piglets withStreptococcus faecalis increased during 24 h the concentration of circulating granulocytes and the concentration of active phagocytes. An even more pronounced effect was obtained when formolizedS. faecalis cells were applied intraperitoneally to germfree piglets. This treatment elevated the phagocytosis index also in conventional piglets, as well as in germfree piglets previously given cyclophosphamide.Escherichia coli O 83 or a mixture of anaerobic bacteria did not cause any serious changes in the activity of phagocytosis in gnotobiotic piglets.S. faecalis seems to be a natural factor stimulating both the release of granulocytes from their depots as well as their phagocytic activity.     

Low-dose intragastric administration of Phaseolus vulgaris agglutinin (PHA) does not induce immunoglobulin E (IgE) production in Sprague-Dawley rats

Native Phaseolus vulgaris agglutinin (PHA) poses a potential health threat, when ingested with improperly cooked red kidney beans. Since PHA triggers human basophilic granulocytes in culture to rapidly release considerable amounts of interleukin-(IL-)4 and IL-13, key cytokines for inducing immunoglobulin E (IgE) production, the question was addressed whether this lectin can evoke in vivo IgE production. IgE-low-responder (Sprague-Dawley) rats received PHA (6 mg/rat/day) intragastrically by gavage over a period of 10 days. Up to day 35, there was no IgE induction regardless of whether the animals were boostered subcutaneously with PHA or not, indicating that PHA cannot be regarded as a general IgE inducer in rats.     

Differential binding of lectins to haemocytes of the mussel Mytilus edulis

Summary Pre- and post-embedding techniques were used to investigate the ultrastructural binding of a range of lectins to the haemocytes of the mussel Mytilus edulis. Direct and indirect labelling procedures were employed using colloidal gold and ferritin-labelled lectins, or biotinylated lectins followed by gold-labelled streptavidin. Cell surface receptors were present for lectins from Helix pomatia (HPA), Helix aspersa (HAA), Triticum vulgaris (WGA) and Tetragonolobus purpureas (TPA). Double labelling of haemocytes with HPA and WGA demonstrated binding sites for both lectins on the plasma membrane of the majority of haemocytes. Endocytosis of colloidal gold-labelled HPA was observed for unfixed haemocytes. Three classes of haemocyte were identified by use of morphological criteria: hyalinocytes; granulocytes containing small granules; and granulocytes containing large granules. Lectin binding showed the small granules of the granulocytes to be HPA-positive and the large granules of the granulocytes to be WGA-positive. The WGA-positive granules demonstrated a differential pattern of binding according to granule size. Binding sites for the lectin from Arachis hypogaea (PNA) were not demonstrated on the cell surface, but did show an affinity for the heterochromatin region of the nucleus in post-embedding protocols.     

Blood morphology of the European eel (Anguilla anguilla). II. Studies on granulopoiesis]

It was found that the granulocytes for Anguilla anguilla mainly form in the kidney. The stem cell of granulocytes is the large hemocytoblast. Sequences of development were established for the heterophil, cosinophil and basophil granulocytes. Myeloblasts, promyelocytes, metamyelocytes, mature red-shaped and segmented forms were described as stages of development of the heterophil granulocytes. In the case of the eosinophil and basophil granulocytes the myeloblastic and myelocytic stages could be demonstrated as well as the mature granulocyte. By the aid of special granular staining, phase contrast observations, supravital and cytochemical investigations the granulocytes could be described. The cytochemical proof for the granulocytes delivered a distribution pattern of the lipids, carbohydrates, RNA (RNS) and enzymes: unspecific esterase, alkaline and acid phosphatases, oxydase and peroxydase. By means of heart puncture a larger loss of blood was caused and the hematopoiesis stimulated. The following blood letting gave indications of how rapidly the granulopoiesis develops and how much time maturing takes. After a larger loss of blood the new-growth of the granulocytes will be completed nine days later.     

Characterization of monoamine oxidase activity present in human granulocytes and lymphocytes

The characterization of monoamine oxidase (MAO) activity in lymphocytes and granulocytes was studied by using cells prepared from human blood. The specific activities of the enzyme towards beta-phenylethylamine (PEA), benzylamine (Bz), tyramine (TYR) and 5-hydroxytryptamine (5-HT) were found to be 5-times higher in lymphocytes than in granulocytes. The absence of the semicarbazide-sensitive amine oxidase (SSAO) was confirmed by the lack of effect of semicarbazide on the benzylamine oxidation. The presence of MAO-B was corroborated by the inhibition of PEA oxidation with nanomolar deprenyl concentrations and by inhibition of TYR oxidation with high clorgyline concentrations, as well as by the simple sigmoid curve obtained in both cases. These results, together with the substrate preferences, suggest that the MAO activity of human granulocytes and lymphocytes is predominantly of the B form. For each fraction the kinetic constants were determined towards PEA, TYR and Bz as substrates. The Km values were similar for both cellular samples, whereas the Vmax values were higher in lymphocytes than in granulocytes. MAO-B was titrated with [3H]pargyline in order to find out the number of active sites. The corresponding molecular concentration, Kcat values and turnover number showed the presence of related enzymes in human granulocytes and lymphocytes.     

Endocytosis and intracellular degradation of heterologous protein by eosinophilic granulocytes isolated from rainbow trout (Oncorhynchus mykiss) posterior intestine

Summry— Adherence capacity to tissue substrate, endocytosis capacity for heterologous proteins, and proteolytic activity were determined in intestinal granulocytes (EGCs) isolated from healthy adult rainbow trout. The percentage of cells that could adhere to a smooth plastic surface increased with increasing incubation time. Endocytosis was effective for heterologous (human immunoglobulin G, IgGh; ovine somatotropin, oST) but not homologous proteins (recombinant trout somatotropin, rtST). The activity of cathepsin D increased significantly after the endocytosis of a heterologous protein. Finaly, the analysis of immunoblots of homogenates of granulocytes incubated in the presence of the two different proteins was used to show the endocytosis and degradation of heterologous proteins. These results show that isolated EGCs can endocytose and degrade heterologous proteins.     

Aloe-emodin-induced DNA fragmentation in the mouse in vivo comet assay

Aloe-emodin (AE) and derivatives may be present as undesired components co-extracted during extraction of plants containing anthraquinonic derivatives for preparation of diacetylrhein. AE is a well-known in vitro mutagen, but up to now it failed to induce any clear in vivo genotoxic activity in the chromosome aberration assay in rat bone marrow or the in vivo/in vitro UDS test in liver. However, the two target organs noted during rodent carcinogenicity studies with danthron and emodin, two other well-known anthraquinone derivatives, are the colon and the kidney. Therefore, the choice of the organs for testing the genotoxicity of AE, i.e. bone marrow and liver, may be considered inadequate to demonstrate a possible in vivo genotoxic activity. In this context, the in vivo mouse comet assay was performed on both isolated kidney and colon cells in order to demonstrate a possible organospecific genotoxicity after oral administration of AE. Concurrently, the Ames test and the in vitro micronucleus assay with TK6 human lymphoblastoid cells were performed in their microscale version both with S9 from Aroclor 1254-induced liver or kidney, and without S9.AE induced primary DNA damage in the liver and in the kidney as observed between 3 and 6 h after two oral administrations at 500, 1000 and 2000 mg/kg bw, underlining an in vivo genotoxic mechanism of action. Furthermore, AE induced a clear genotoxic activity both in the Salmonella typhimurium strains TA1537 and TA98 and in the in vitro micronucleus assay in the absence as well as in the presence of metabolic activation. As no significant variation in the genotoxic activity of AE was noted when using either liver or kidney S9-mix, it seems that no quantitatively and/or qualitatively specific renal metabolism occurs. The kidney may be a target organ of AE as it is the major route of excretion. Under such conditions the separation of AE components should take place and the residual content of undesired AE derivatives should be made as low as reasonably achievable. AE present in plant extracts should be considered as an in vivo genotoxin and this property should be taken into account in the risk assessment for human exposure.     

Renal cortex guanylate cyclase. Preferential enrichment in glomerular membranes.

1. The localisation and some of the properties of rabbit kidney cortex guanylate cyclase (GTP pyrophosphatase lyase (cyclizing) EC 4.6.1.2) have been studied. Upon fractionation of dissociated renal cortex, guanylate cyclase activity was preferentially enriched in fractions of pure glomeruli, where its specific activity was 44.5 times that measured in tubular fragments. Most, if not all, of the glomerular activity was found to be firmly membrane-bound, whereas the guanylate cyclase activity of the tubules was mainly soluble. Therefore, particulate guanylate cyclase activity could serve as marker enzyme for kidney glomeruli. 2. All hormones or hormone-like agents tested were without effect on kidney guanylate cyclase activity. Triton X-100 stimulated both glomerular and tubular activity. 3. Considering the high cyclic GMP forming capacity of kidney glomeruli, part of the cyclic GMP found in urine might be synthetized locally in these structures.