Kallikrein-related Peptidase 5 (KLK5) Expression and Distribution in Canine Cutaneous Squamous Cell Carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is one of the most common types of malignant skin cancer in dogs, representing 3.9-10.4% of all canine skin tumours. Although the metastatic potential of cSCC is debated, it appears to mimic that observed in man. In man, predictive histopathological features for metastasis include tumour depth, lesions >5-6 mm in depth, and invasion of muscle, cartilage or bone. In dogs, some reports have focused on the clinical features and long-term progression of cSCC, but a gold standard treatment has not yet been developed. We explored the protein expression of kallikrein-related peptidase 5 (KLK5), an important modulator of skin homeostasis, in normal canine skin and in examples of cSCC. KLK5 was highly expressed in the upper stratum granulosum, stratum corneum, hair follicles and sweat glands, skin sites where human KLK5 has been shown to be involved in physiological processes including keratinocyte desquamation, antimicrobial defence, lipid permeability and pigmentation. In cSCC, tumour cells at the deep margin, as well as those in the centre of keratin pearls, displayed cytoplasmic expression of KLK5. Some of the KLK5 immunoreactive cells also expressed vimentin, suggesting that they may be undergoing epithelial-mesenchymal transition and therefore have a more invasive behaviour than those expressing only KLK5. KLK5 may be a novel molecular biomarker useful for predicting prognosis of cSSC in dogs.

New potential targets to modulate neutrophil function in inflammation.

The importance of neutrophils in human disease such as rheumatoid arthritis, asthma, adult respiratory distress syndrome, and COPD has prompted the search for drugs capable to slow down neutrophil-dependent inflammation, without interference with innate immune responses. In this review, we summarize new potential drugs targets against neutrophils mediated inflammatory responses.

Development and growth of digestive system organs of European and Japanese quail at 14 days post-hatch.

The objective of this study was to evaluate the development and growth of the digestive system organs, from the 11th day of incubation until the 14 d post-hatch in European and Japanese quail. On days 11, 13 and 15 of incubation at hatch and at 4, 7, 10 and 14 d post-hatch, embryos or chicks of European and Japanese quail were analyzed. After 15 d of incubation, samples from stomach and small intestine were analyzed by microscopy. European quail had significantly heavier body weight at 15 d of incubation and after 4 d post-hatch. The digestive system weight progressively increased with age and was similar between European and Japanese quail at 11, 13, and 15 d of incubation and 10 d post-hatch, while relative weight of digestive system was similar between quail type with great values at 4 d post-hatch. For relative weight of the small intestine + pancreas, the weight of the proventriculus and of the gastric ventricle increased significant by among ages analyzed in both types of quail. At hatch, proventriculus had functional secretory cells and mucosa of gastric ventricle had a thin coilin membrane. In small intestine segments, at 15 d of incubation the height of the villi was similar among duodenum, jejunum, and ileum (80 µm). Villi had elongated shape towards the intestinal lumen, covered by enterocytes and dispersed goblet cells with PAS+ and AB+ contend in all segments. The number of goblet cell/villi increased in segments until 7 to 10 d post-hatch. Duodenum increases the villi up to 14 d, while the jejunum and ileum up to 10 and 4 d, respectively. Based on our data in digestive system growth, a shorter period of post-hatch fast and specific diets to quail during first days of growth is recommended to both quail types. It is concluded that the development and growth of different organs of the digestive system up to 14 d of age was similar between European and Japanese quail.

Activation of kinin B receptor triggers differentiation of cultured human keratinocytes.

BACKGROUND: Keratinocyte life span is modulated by receptors that control proliferation and differentiation, key processes during cutaneous tissue repair. The kinin B(1) receptor (B(1)R) has been reported in normal and pathological human skin, but so far there is no information about its role in keratinocyte biology. OBJECTIVES: To determine the consequence of kinin B(1)R stimulation on tyrosine phosphorylation, a key signalling mechanism involved in keratinocyte proliferation and differentiation. METHODS: Subconfluent primary cultures of human keratinocytes were used to investigate tyrosine phosphorylation, epidermal growth factor receptor (EGFR) transactivation, cell proliferation and keratinocyte differentiation. Cell proliferation was assessed by measuring bromodeoxyuridine incorporation whereas assessment of cell differentiation was based on the expression of filaggrin, cytokeratin 10 (CK10) and involucrin. RESULTS: The major proteins phosphorylated, after B(1)R stimulation, were of molecular mass 170, 125, 89 and 70 kDa. The 170- and 125-kDa proteins were identified as EGFR and p125(FAK), respectively. Phosphorylation was greatly reduced by GF109203X and by overexposure of keratinocytes to phorbol 12-myristate 13-acetate, indicating the participation of protein kinase C. B(1)R stimulation did not increase [Ca(2+)]i, but triggered EGFR transactivation, an event that involved phosphorylation of Tyr(845), Tyr(992) and Tyr(1068) of EGFR. B(1)R stimulation did not elicit keratinocyte proliferation, but triggered cell differentiation, visualized as an increase of filaggrin, CK10 and involucrin. Blockade of EGFR tyrosine kinase by AG1478, before B(1)R stimulation, produced an additional increase in filaggrin expression. CONCLUSIONS: The kinin B(1)R may contribute to keratinocyte differentiation and migration by triggering specific tyrosine signalling pathways or by interacting with the ErbB receptor family.

Fatty and hydroxycarboxylic acid receptors: The missing link of immune response and metabolism in cattle.

Fatty and hydroxycarboxylic acids are one of the main intermediates of energy metabolism in ruminants and critical in the milk production of cattle. High production demands on a dairy farm can induce nutritional imbalances and metabolism disorders, which have been widely associated with the onset of sterile inflammatory processes and increased susceptibility to infections. The literature suggests that short-chain fatty acids (SCFA), long-chain fatty acids (LCFA) and hydroxycarboxylic acids are relevant modulators of the host innate inflammatory response. For instance, increased SCFA and lactate levels are associated with subacute ruminal acidosis (SARA) and the activation of pro-inflammatory processes mediated by diverse leukocyte and vascular endothelial cells. As such, free LCFA and the ketone body ß-hydroxybutyrate are significantly increased in the plasma 1-2 weeks postpartum, coinciding with the time period in which cows are more susceptible to acquiring infectious diseases that the host innate immune system should actively oppose. Today, many of these pro-inflammatory responses can be related to the activation of specific G protein-coupled receptors, including GPR41/FFA3 and GPR43/FFA2 for SCFA; GPR40/FFA1 and GPR120/FFA4 for LCFA, GPR109A/HCA2 for ketone body ß-hydroxybutyrate, and GPR81/HCA1 for lactate, all expressed in different bovine tissues. The activation of these receptors modulates the release of intracellular granules [e.g., metalloproteinase-9 (MMP-9) and lactoferrin], radical oxygen species (ROS) production, chemotaxis, and the production of relevant pro-inflammatory mediators. The article aimed to review the role of natural ligands and receptors and the resulting impact on the host innate immune reaction of cattle and, further, to address the most recent evidence supporting a potential connection to metabolic disorders.

Activation of kinin B1 receptors induces chemotaxis of human neutrophils.

Kinins are biologically active peptides that are powerful mediators of cellular inflammation. They mimic the cardinal signs of inflammation by inducing vasodilatation and by increasing vascular permeability and pain. Neutrophils are chemoattracted to sites of inflammation by several stimuli. However, the evidence concerning the chemotactic effect of kinin peptides has been contradictory. We analyzed the chemotactic effect of kinin B(1) receptor agonists on neutrophils isolated from peripheral blood of human healthy subjects. Chemotaxis was performed using the migration under agarose technique. To test the effect of B(1) receptor agonists, each assay was carried out overnight at 37 degrees C in 5% CO(2)-95% air on neutrophils primed with 1 ng/ml interleukin-1beta. Simultaneous experiments were performed using unprimed cells or cells challenged with formyl-Met-Leu-Phe (fMLP). A clear chemotactic activity was observed when primed neutrophils were challenged with Lys-des[Arg(9)]-bradykinin (LDBK) or des[Arg(9)]-bradykinin at 10(-10) M but not when unprimed cells were used. A reduction in the chemotactic response was observed after priming of cells in the presence of 0.5 mM cycloheximide and 10 mug/ml brefeldin A, suggesting that some protein biosynthesis is required. Techniques such as reverse transcriptase-polymerase chain reaction and in situ hybridization confirmed the expression of the B(1) receptor mRNA, and immunocytochemistry and autoradiography demonstrated the expression of the B(1) receptor protein. In contrast to other chemoattractants such as fMLP, cytosolic intracellular calcium did not increase in response to the B(1) receptor agonist LDBK. A generation of kinin B(1) receptor agonists during the early phase of acute inflammation may favor the recruitment of neutrophils to the inflammatory site.

The calcium-activated potassium channel KCa3.1 plays a central role in the chemotactic response of mammalian neutrophils.

AIM: Neutrophils are the first cells to arrive at sites of injury. Nevertheless, many inflammatory diseases are characterized by an uncontrolled infiltration and action of these cells. Cell migration depends on volume changes that are governed by ion channel activity, but potassium channels in neutrophil have not been clearly identified. We aim to test whether KCa3.1 participates in neutrophil migration and other relevant functions of the cell. METHODS: Cytometer and confocal measurements to determine changes in cell volume were used. Cells isolated from human, mouse and horse were tested for KCa3.1-dependent chemotaxis. Chemokinetics, calcium handling and release of reactive oxygen species were measured to determine the role of KCa3.1 in those processes. A mouse model was used to test for neutrophil recruitment after acute lung injury in vivo. RESULTS: We show for the first time that KCa3.1 is expressed in mammalian neutrophils. When the channel is inhibited by a pharmacological blocker or by genetic silencing, it profoundly affects cell volume regulation, and chemotactic and chemokinetic properties of the cells. We also demonstrated that pharmacological inhibition of KCa3.1 did not affect calcium entry or reactive oxygen species production in neutrophils. Using a mouse model of acute lung injury, we observed that Kca3.1(-/-) mice are significantly less effective at recruiting neutrophils into the site of inflammation. CONCLUSIONS: These results demonstrate that KCa3.1 channels are key actors in the migration capacity of neutrophils, and its inhibition did not affect other relevant cellular functions.

Butyric acid stimulates bovine neutrophil functions and potentiates the effect of platelet activating factor.

Increased short-chain fatty acid (SCFA) production is associated with subacute ruminal acidosis (SARA) and activation of inflammatory processes. In humans and rodents, SCFAs modulate inflammatory responses in the gut via free fatty acid receptor 2 (FFA2). In bovines, butyric acid is one of the most potent FFA2 agonists. Its expression in bovine neutrophils has recently been demonstrated, suggesting a role in innate immune response in cattle. This study aimed to evaluate if butyric acid modulates oxidative and non-oxidative functions or if it can potentiate other inflammatory mediators in bovine neutrophils. Our results showed that butyric acid can activate bovine neutrophils, inducing calcium (Ca(2+)) influx and mitogen-activated protein kinase (MAPK) phosphorylation, two second messengers involved in FFA2 activation. Ca(2+) influx induced by butyric acid was dependent on the extracellular and intracellular Ca(2+) source and phospholipase C (PLC) activation. Butyric acid alone had no significant effect on reactive oxygen species (ROS) production and chemotaxis; however, a priming effect on platelet-activating factor (PAF), a potent inflammatory mediator, was observed. Butyric acid increased CD63 expression and induced the release of neutrophil granule markers matrix metalloproteinase-9 (MMP-9) and lactoferrin. Finally, we observed that butyric acid induced neutrophil extracellular trap (NET) formation without affecting cellular viability. These findings suggest that butyric acid, a component of the ruminal fermentative process, can modulate the innate immune response of ruminants.

Immunolocalization of bradykinin B2 receptors on skeletal muscle cells.

The kallikrein-kinin system has been implicated in the inflammatory process, blood pressure regulation, renal homeostasis, and glucose utilization. The effects of kallikrein and kinin on glucose uptake by the skeletal muscle are well established; however, the occurrence and the cellular distribution of the kinin receptor(s) mediating these effects in the striated muscle are unknown. Using anti-peptide antibodies raised against the predicted intra- and extracellular domains of the B2 receptor and the peroxidase/antiperoxidase system, we have been able to detect the B2 receptor on the plasma membrane of striated skeletal muscle cells of the rat hindlimb. A strong immunostaining appeared as a rim of immunoreactive material located on the periphery of striated muscle cells. Cross-sectioned and longitudinally sectioned cells revealed a similar staining pattern. Alternatively, the immunostaining with specific antibodies to tissue kallikrein and to T-kininogen did not yield a significant staining of the striated muscle cells. Localization of the B2 receptor on the surface of striated muscle cells provides a structural basis for the hypothesized physiological functions of the kinin system in the skeletal muscle.

Physical interaction between Langerhans cells and T-lymphocytes during antigen presentation in vitro.

Physical interaction between Langerhans cells and T cells is an essential requirement for antigen presentation. In this study we report the ultrastructural characteristics of the antigen-specific physical interactions that occur in vitro between murine Langerhans cells and T cells. Epidermal Langerhans cells enriched to 78% purity by a panning method were pulsed with 2,4-dinitrophenyl-Limulus polyphemus hemocyanin and co-incubated with syngeneic T lymphocytes primed in vivo with the same antigen. A substantial number of conjugates constituted by Langerhans cells surrounded by three or more lymphocytes were obtained after 60 min of incubation at 4 degrees C. Electron microscopy of the conjugates revealed that Langerhans cells and T lymphocytes interacted by two type of contacts. Type I was characterized by glycocalyx-glycocalyx interaction that occurred in relation to protrusions or microvilli of both cells. Type II was characterized by wide and tight areas of close apposition between Langerhans cells and T-lymphocyte plasma membranes. In these areas there were zones with intercellular bridges and small septilaminar junctions highly suggestive of gap junctions. An electron-immunogold procedure demonstrated the presence of DNP-LPH antigen on the type I contact. Our findings suggest that type I contact may represent the locus for antigen presentation whereas the type II contact may be involved in keeping adhesiveness between Langerhans cells and lymphocytes during antigen presentation.

Alpha 1-antitrypsin expression in human thyroid papillary carcinoma.

Alpha 1-antitrypsin is a plasma serine protease inhibitor originally used as a marker for tumors of histiocytic origin. Our casual finding of immunoreactive alpha 1-antitrypsin in one case of thyroid papillary carcinoma led us to investigate its presence in 10 thyroid papillary carcinomas by applying immunocytochemical and immunochemical techniques to tissue sections and Western blots of tissue homogenates prepared from neoplastic tissue and from uninvolved normal areas in the vicinity of each tumor. The immunocytochemical study was performed in both thyroid tissue and metastatic regional lymph nodes. This analysis revealed immunoreactivity for alpha 1-antitrypsin in nine of the 10 cases studied. Immunoreactivity was intense in some of the cells forming the papillar and follicular structures. These cells were intermingled with completely unstained tumoral cells. In contrast to neoplastic tissue, the normal thyroid tissue present in the vicinity of each tumor showed no staining for alpha 1-antitrypsin. The electrophoretic analysis performed on homogenates prepared from both tumoral and normal thyroid tissue revealed a drastic reduction in the band corresponding to thyroglobulin in the tumoral tissue compared with normal thyroid extracts, where it represented the major protein. Western blotting and immunoprinting with a polyclonal alpha 1-antitrypsin antibody confirmed the results obtained with immunocytochemistry about the presence of this protease inhibitor in neoplastic thyroid tissue. Immunoprinting with the anti-alpha 1-antitrypsin antibody revealed an intense immunoreactive band of 53 kDa in the extracts prepared from tumoral tissue. This band had exactly the same apparent molecular mass previously described by others for alpha 1-antitrypsin purified from plasma and was identical to the molecular mass of the purified commercial standard employed.

Immunoreactive kallikrein localization in the rat kidney: an immuno-electron-microscopic study.

The cellular and subcellular localization of immunoreactive kallikrein was studied in the rat kidney using the peroxidase-antiperoxidase (PAP) method for the electron microscope. The effect of various tissue-processing protocols on ultrastructural preservation and immunocytochemical staining was evaluated by fixing kidneys with four different mixtures. The tissues were immunostained and further stained with OsO4 or silver methenamine. The best ultrastructural and immunocytochemical staining was obtained with Zamboni's-glutaraldehyde fixative. The kallikrein-immunoreactive cell type was identified, according to its localization and ultrastructural features, as the connecting-tubule cell. Immunoreactive kallikrein was concentrated mainly in the upper one-third of the cell and at both sides of the nuclei, and to a less extent was associated with the plasma membranes and basolateral infoldings. The immunoreactivity was related to free polyribosomes, the rough endoplasmic reticulum (RER), and the Golgi complex, suggesting that kallikrein is actively synthesized in this particular type of cell.

Probing for the bradykinin B2 receptor in rat kidney by anti-peptide and anti-ligand antibodies.

The kallikrein-kinin system is involved in the inflammatory process, in blood pressure regulation, and in renal homeostasis. The presence of kallikreins, kininogens, and kinins in renal tissues and fluids is well established; however, the occurrence and distribution of the bradykinin (B2) receptor in the kidney are unknown. Using chemically cross-linked conjugates of bovine serum albumin and the B2 agonist bradykinin or the potent B2 antagonist HOE140, followed by antibodies to the respective ligand and the peroxidase-anti-peroxidase system, we were able to detect the B2 receptor. The receptor has been found in straight portions of the proximal tubules, in distal straight tubules, in connecting tubules, and in collecting ducts of rat kidney. The staining patterns produced by the ligand conjugate-antiligand approach are in agreement with those obtained by conventional autoradiography using [125I]-Tyr0-bradykinin. Immunocytochemical localization of B2 receptor by antipeptide antibodies to the receptor confirmed these findings and demonstrated the presence of B2 receptor in the basal infoldings and luminal membranes of the tubule cells, and in smooth muscle cells of the cortical radial artery and of afferent arterioles. Co-localization of the B2 receptor with kallikrein and kininogens in connecting tubule cell and collecting duct cell layers, respectively, provides a structural basis for the hypothesized physiological functions of the kallikrein-kinin system in the kidney.

Tissue kallikrein is associated with prolactin-secreting cells within human growth hormone-secreting adenomas.

Tissue kallikrein is a serine protease which may be involved in the intracellular processing of prolactin in the anterior pituitary gland. The expression of tissue kallikrein, in the rat, is promoted by oestrogen and inhibited by dopamine. Human and rat prolactinomas contain markedly increased amounts of tissue kallikrein; this is comparatively reduced if patients are pretreated with the dopamine agonist, bromocriptine, before surgery. Some GH-secreting adenomas are mixed and also contain prolactin-secreting cells. We therefore investigated 27 GH-immunostaining human pituitary adenomas for the presence of immunoreactive tissue kallikrein. Sixteen of the adenomas had positive immunostaining for prolactin; eight of these patients had associated clinical hyperprolactinaemia before the tumour was removed. Tissue kallikrein immunoreactivity was found in ten adenomas, all of which also had prolactin immunopositivity. There was a close relationship between the percentage of cells staining for prolactin and tissue kallikrein but not for GH. A further eight adenomas had patchy positivity, i.e. less than 1% of cells immunostained for tissue kallikrein and six of these also had some prolactin-staining cells. Nine out of eleven purely GH-staining adenomas had no tissue kallikrein immunopositivity, the remaining two showing patchy staining. A review of bromocriptine responsiveness, as assessed by mean GH hormone levels during oral glucose tolerance tests before and after therapy was commenced, indicated that patients with adenomas which stained for prolactin and tissue kallikrein were more likely to respond to bromocriptine than those which failed to do so.

Characterization of a tissue kallikrein in human prolactin-secreting adenomas.

Immunoreactive tissue kallikrein was co-localized with prolactin in all the eleven prolactin-secreting adenomas of the human anterior pituitary gland examined in this study. The intracellular distribution of immunoreactivity in the prolactin-secreting cells suggests that tissue kallikrein is located within the Golgi complex of these cells. Both the intracellular hormone-processing action and the kininogenase activity of tissue kallikrein may be of functional importance in human prolactinomas.

Anatomical relationship between kallikrein-containing tubules and the juxtaglomerular apparatus in the human kidney.

Current evidence suggests a functional and biochemical link between the renin and the kallikrein systems. The purpose of this work was to study the localization of kallikrein along the human nephron to elucidate whether there exists an anatomical base for such interrelation. Serial sections of human kidney tissue were stained by immunocytochemical methods with antisera against kallikrein. Kallikrein immunostaining was observed exclusively in segments of the distal nephron lying in the cortical labyrinths and forming arcades in its distal portion. Consistently the tubules containing kallikrein established a close anatomical relationship with the afferent arteriole of the juxtaglomerular apparatus providing an anatomical base for an interaction between the renin and kallikrein systems in the human kidney.

Cellular and subcellular distribution of 2,4-dinitrophenyl groups in mouse epidermis and regional lymph nodes after epicutaneous application of 2,4-dinitrofluorobenzene.

The cellular and subcellular distribution of 2,4-dinitrophenyl (DNP) groups in the epidermis and regional lymph nodes of the mouse was investigated after epicutaneous application of 2,4-dinitrofluorobenzene (DNFB) to sensitized and non-sensitized mice. The peroxidase-antiperoxidase method and the immunogold technique were used to visualize the DNP groups at both light and electron microscopic levels. The highest intensity of immunolabelling was found on tonofilaments of keratinocytes present in the upper layers of the epidermis. On the other hand, in vitro experiments showed that DNFB has the capacity to bind keratin which, together with immunocytochemistry, suggests that this molecule may be one of the skin protein carriers for DNFB. In addition, intense immunostaining for DNP was observed in the Golgi area of some epidermal Langerhans cells. Cells immunoreactive to DNP were also observed in the marginal sinus of cervical lymph nodes 6, 12 and 24 h after challenge. Immunoelectron microscopy revealed immunoreactive DNP groups in phagosomes of Langerhans cells at this site. The present findings support the hypothesis that the hapten DNFB penetrates passively into the cytoplasm of Langerhans cells, concentrates in the Golgi area and, during the migration of Langerhans cells to the lymph nodes, it is probably processed in the lysosomes before its presentation to T lymphocytes.

Postnatal maturation of tissue kallikrein-producing cells (connecting tubule cells) in the rat kidney: a morphometric and immunohistochemical study.

The mature, fully differentiated connecting tubule (CNT) cell plays an important role in the regulation of serum potassium levels and synthesizes the enzyme tissue kallikrein, a main component of a renal vasoactive system, the kallikrein-kinin system. To characterize the growth of CNT cells (tissue kallikrein-producing cells), we studied the rat kidney at three different time points of postnatal development: at day 5, day 15, and day 30. The CNT cells were identified on tissue sections by a standardized immunohistochemical procedure. The tissue kallikrein content was determined by radioimmunoassay and the activity of the enzyme in kidney homogenates was measured using a selective synthetic substrate. The number of immunolabeled CNT and CNT cells per cortex area gradually increased from day 5 to day 30. A similar rise in the content and activity of tissue kallikrein was observed when the enzyme levels were determined by radioimmunoassay or by the enzymatic method. In addition, the morphometric analysis showed that the distal end of CNT had larger cells that displayed a more intense tissue kallikrein staining than those present in the proximal end, suggesting that the postnatal development of CNT is induced from its juxtamedullary portion. Our results show that tissue kallikrein expression is very low in the newborn rat, increasing gradually with age to reach adult levels at day 30. This finding, together with the morphometric data, suggests immaturity of CNT cells in newborn rats, a fact that could contribute to explaining the high serum potassium levels reported at this stage. In addition, the contrasting behavior of kallikrein and renin in the postnatal development (kallikrein increasing and renin decreasing) could explain the gradual decrease in renal vascular resistance and increase in renal blood flow observed after birth.

Distribution of bradykinin B2 receptors in target cells of kinin action. Visualization of the receptor protein in A431 cells, neutrophils and kidney sections.

Peptides corresponding to sequences derived from predicted extra- and intracellular loops of the rat bradykinin receptor were analyzed for interspecies homology as well as for matches within the present dataset of protein sequences to provide a theoretical basis for the specific recognition of the native cognate protein by antibodies raised against these antigens. Application of polyclonal antibodies raised against the selected peptides allowed the immunocytochemical localization of the native receptor protein in cells of rat and human origin. The detection of the molecule was achieved by different immunohisto- and immunocytochemical methods in combination with light, fluorescence, confocal optical laser and electron microscopy. These results were compared to localization studies by autoradiography. Distribution and subcellular localization were determined in human neutrophils, human epithelial carcinoma cells (A431) and in rat kidney tissue.

Cellular localization of atrial natriuretic peptide and tissue kallikrein in the human hypothalamus.

In the kidney, renal atrial natriuretic peptide (ANP) is considered to play an important role in water and salt homeostasis. Immunoreactive ANP in the brain of lower invertebrates, such as the rat, has been shown to be localized in the hypothalamus and septum. Several studies have investigated the possibility of a regulatory system in the brain similar to that of the kidney. Since neuronal function is acutely sensitive to disturbances of the intracranial water and salt balance we have attempted to immunolocalize ANP-containing cells in the normal human hypothalamus, using a polyclonal antiserum specific to ANP. Also, we have observed tissue kallikrein (TK), using a polyclonal antiserum specific to TK, in the same areas as ANP. A regulatory role for TK on prolactin has been suggested as the rationale for the co-localization of these two hormones in human prolactinomas. Therefore, it could be suggested that TK plays a similar role in the processing of precursor ANP in the brain. It is contemplated to examine the status of these peptides in patients with cerebral oedema.