Regulation Of Fluid Transport Research Articles

Expression of aquaporin 4 in the chicken ovary in relation to follicle development.

In the mammalian ovary, aquaporins (AQPs) are thought to be involved in the regulation of fluid transport within the follicular wall and antrum formation. Data concerning the AQPs in the avian ovary is very limited. Therefore, the present study was designed to examine whether the AQP4 is present in the chicken ovary, and if so, what is its distribution in the ovarian compartment of the laying hen. Localization of AQP4 in the ovarian follicles at different stage of development was also investigated. After decapitation of hens the stroma with primordial follicles and white (1-4mm), yellowish (4-8mm), small yellow and the three largest yellow pre-ovulatory follicles F3-F1 (F3<F2<F1; 20-36mm) were isolated from the ovary. The granulosa and theca layers were separated from the pre-ovulatory follicles. The AQP4 mRNA and protein were detected in all examined ovarian compartments by the real-time PCR and Western blot analyses, respectively. The relative expression of AQP4 was depended on follicular size and the layer of follicular wall. It was the lowest in the granulosa layer of pre-ovulatory follicles and the highest in the ovarian stroma as well as white and yellowish follicles. Along with approaching of the largest follicle to ovulation the gradual decrease in AQP4 protein level in the granulosa layer was observed. Immunoreactivity for AQP4 was present in the granulosa and theca cells (theca interna≥theca externa>granulosa). The obtained results suggest that AQP4 may take part in the regulation of water transport required for follicle development in the chicken ovary.

Characterization of CFTR Activators and Inhibitors by the use of a Planar Patch Clamp System

Cystic fibrosis is caused by malfunction of the chloride channel Cystic Fibrosis Transmembrane Regulator (CFTR). CFTR is expressed in the apical membrane of epithelial cells where it is involved in the regulation of fluid transport across the epithelium. A large number of mutations in the protein are known to cause CFTR to become dysfunctional and only a few pharmaceutical compounds have been developed to treat the disease by restoring the chloride conductance of the channel (1). CFTR is activated by cAMP dependent phosphorylation and is gated by ATP. Activation is typically achieved using forskolin that activates adenylate cyclase which then leads to phosphorylation of the channel via protein kinase A (PKA). Screening for CFTR activators is usually done in the absence of forskolin, but in the presence of low concentrations of cAMP. Studies in animal models support the development of CFTR inhibitors for antisecretory therapy of enterotoxin-mediated diarrheas and polycystic kidney disease. For this purpose an alternative approach can be used. In the presence of internal fluoride CFTR is activated as to the same degree as after forskolin activation. We here present data from Nanion's SyncroPatch 384PE where we activate the channel with either forskolin under fluoride free conditions or by internal solution exchange where the fluoride is washed into the intracellular solution. Our results show that the activation of CFTR by either forskolin or intracellular F- is sensitive to glibenclamide and CFTRInh-172 in a dose - and/or voltage dependent manner. Taken together, these experiments show a stable and cost optimized approach to study the pharmacology of CFTR at high throughput that might empowers new ways in the drug discovery on CFTR.

Adhesion in mammary development: novel roles for E-cadherin in individual and collective cell migration.

Epithelial tissues are essential for barrier function, secretion, and regulation of fluid transport. Their function requires cell polarity and cell-cell adhesion, mediated through intercellular junctions. Conversely, disruption of adhesion and polarity is thought to drive cancer progression. The mammary gland is an important model for cell adhesion due to its postnatal hormonally regulated development; ducts undergo branching morphogenesis in response to steroid hormones during puberty. These hormonal signals induce a transition from simple to stratified architecture, initiated by asymmetric luminal cell divisions. Ductal elongation is accomplished by this multilayered, low-polarity epithelium, and polarity is reestablished as elongation ceases. The requirement for cell adhesion has been tested in 3D culture and in vivo, using gene deletion, knockdown, and misexpression in both developmental and homeostatic contexts. Attention has focused on E-cadherin, the major classical cadherin in luminal epithelial cells. Classic studies revealed a requirement for E-cadherin during lactation, and E-cadherin loss is widely posited to promote metastasis. However, recent findings demonstrated a broader requirement for E-cadherin during branching morphogenesis and homeostasis and also, surprisingly, in epithelial dissemination. These studies suggest that long-standing models of the role of adhesion in epithelial biology need to be revisited. Advances in inducible gene expression and knockdown, CRISPR/Cas9 technology, and fluorescent labeling of genetically modified cells offer the opportunity to test the roles of diverse adhesion systems and to develop a mechanistic understanding of how cell adhesion regulates development and cancer.

Fusion Dependent Activation of Vesicular P2X4 Receptors Leads to a Volume Increase in Alveolar Type II Epithelial Cells - Coupling Secretion and Lung Fluid Homeostasis?

Regulated fluid transport in the lung is a necessity for maintenance of a healthy lung environment. However, the role of the alveolar epithelium, consisting of alveolar type I and II (ATII) cells, remains largely undetermined. While the ATII cell is mainly known for secretion of lung surfactant via exocytosis of lamellar bodies (LBs), it is also thought that it may assist in fluid homeostasis as well, both critical for alveolar function. Directed ion transport across the ATII cell will result in secretion or absorption of fluid into or from the alveolar hypophase, respectively. We have recently described that exocytic fusion of LBs with the plasma membrane (PM) results in a transient, non-selective, inward-rectifying, cation current across P2X4 purinergic receptors located on the membranes of fused LBs (PNAS 2011, 108(35):14503-8). This leads us to propose that regulation of LB exocytosis also modulates fluid transport across the alveolar epithelium via this “fusion-activated” cation influx. Experiments combining fluorescence and atomic force microscopy (AFM) confirmed that exocytosis of LBs leads to an instant increase in ATII cell volume that is regulated within mins. Following stimulation of ATII cells with ATP, the height of the ATII cells increased by 30% in cells when LBs fused with the PM after stimulation, but slightly decreased in cell without fusing LBs. These data indicate a link between LB fusion (and hence surfactant secretion) and regulation of fluid transport.

The Role of the NHERF Family of PDZ Scaffolding Proteins in the Regulation of Salt and Water Transport

The four members of the NHERF (Na(+)/H(+) exchanger regulatory factor) family of PDZ adapter proteins bind to a variety of membrane transporters and receptors and modulate membrane expression, mobility, interaction with other proteins, and the formation of signaling complexes. All four family members are expressed in the intestine. The CFTR (cystic fibrosis transmembrane regulator) anion channel and the Na(+)/H(+) exchanger NHE3 (Na/H exchanger- isoform 3) are two prominent binding partners to this PDZ-adapter family, which are also known key players in the regulation of intestinal electrolyte and fluid transport. Experiments in heterologous expression systems have provided a number of mechanistic models how NHERF protein interactions can affect the function of their targets at the molecular level. Recently, NHERF1, 2, and 3 knockout mice have become available, and this review summarizes the reports on electrolyte and fluid transport regulation in the native intestine of these mice.

Hypertonic saline inhibits luminal sodium channels in respiratory epithelium

Physical exercise with increased ventilation leads to a considerable rise in water loss from the airways. The mechanisms underlying the regulation of transepithelial fluid transport necessary to compensate for these losses are unknown but may include changes in luminal ion channel conductance. The present study was designed to examine the effects of an increase in luminal chloride and sodium concentrations which may locally occur during hyperventilation on luminal ion conductance in the respiratory epithelium of healthy controls and patients diagnosed with cystic fibrosis (CF). Changes in luminal chloride and sodium conductance were inferred by recording nasal potential difference in eight healthy subjects and 10 patients with CF, using superfusing solutions based on isotonic saline (150 mM) on one occasion and solutions based on hypertonic saline (300 mM) on the other. Switching from isotonic to hypertonic saline superfusion decreased potential difference in controls and CF patients significantly. Amiloride induced a decrease of potential difference which was larger with isotonic than with hypertonic saline (controls 9.5 +/- 6.1 vs. 3.7 +/- 4.6 mV; CF 17.2 +/- 7.2 vs. 9.8 +/- 7.6 mV). Chloride conductance stimulated with solutions low in chloride and containing isoproterenol was not significantly changed by hypertonic saline solutions compared with isotonic solutions in both groups. The findings indicate a significant inhibition of luminal sodium conductance by high luminal sodium concentrations. This mechanism may be involved in the regulation of fluid transport across the respiratory epithelium during exercise and in the improvement of mucociliary clearance and lung functions with inhalation of hypertonic saline in CF.

Ion and Fluid Transport Properties of Small Airways in Cystic Fibrosis

Small airways constitute a major site of pathology in cystic fibrosis (CF) and provide most of the surface area of the conducting airways of the lung. Little is known, however, about the impact of CF on ion and fluid transport in small (bronchiolar) airways. To describe the ion and fluid transport properties of CF bronchiolar epithelium. Primary cultures of human bronchial and bronchiolar (non-CF and CF) epithelial cells were obtained. The bioelectric properties were studied in Ussing chambers and the airway surface liquid (ASL) height was measured with confocal microscopy. Primary cultures of DeltaF508 CF bronchiolar epithelial cells displayed higher transepithelial resistance than non-CF cultures, whereas baseline short circuit current and amiloride-inhibitable short circuit current were similar in both preparations. The ASL height was significantly smaller in CF compared with non-CF preparations. In the presence of amiloride, addition of forskolin increased short circuit current in non-CF but not in CF bronchiolar cultures, and the ATP-induced increase in short circuit current was lower in CF than in non-CF cultures. Non-CF bronchiolar preparations displayed larger short circuit current and fluid secretion in responses to forskolin than non-CF bronchial preparations, suggesting that CFTR-dependent Cl(-) transport may play a more important role in the regulation of fluid transport in small airways than in large airways. In CF small airways, defective Cl(-) secretion combined with unregulated (persistent) Na(+) absorption results in ASLdepletion.

Monoamine receptors in human corneal epithelium and endothelium

Monoamine receptors are found throughout the body. Reports about the presence of monoamine receptors in the human cornea are inconsistent. Immunohistochemistry, immunofluorescence and immunoblotting were used to localize monoamine receptor sites on human corneal epithelium and endothelium. Antibodies to alpha-1, beta-1 and beta-2 adrenergic receptors and to D1-like and 5HT-7 receptors were bound in corneal epithelium. Antibodies to alpha-1, alpha-2A, beta-1 and beta-2 adrenergic receptors and to 5HT-7 receptors were bound in corneal endothelium. Our data demonstrate the presence of several monoamine receptors in the human cornea. These receptors may play a role in the regulation of fluid transport or corneal homeostasis.

Immunohistology of aquaporin-1 and stem cell factor-receptor in human undescended testes.

Both aquaporin (AQP) 1 and the stem-cell factor/C-kit system seem to have a definite role in testis function, but very few studies have been reported in humans, especially in the paediatric age group. With the present study we wanted to investigate the expression of these proteins to better delineate their role in normal and pathologic testes. Immunohistology using AQP 1 and C-kit antibodies was performed on paraffin sections of open-testicular biopsies from 32 undescended testes. The testes of cryptorchid patients, with ages ranging from 2 to 15 years, were biopsied during an orchidopexy operation, after obtaining informed consent. Control biopsies, from 8 patients of matched age, were obtained during operations for inguinal hernia or hydrocele, always after obtaining informed consent. Positive results were recorded as diffuse or focal patterns and scored as weak, moderate or strong immunostaining. AQP 1 antibody strongly depicted microvessel endothelial cells, but was unlabeled in endotubular and interstitial cell lines, in both control and undescended testes. The C-kit immunostaining in normal testes revealed a diffuse, strong staining in the cytoplasm of spermatogonia and primary spermatocytes. However, in the undescended testes a focal C-kit immunolabelling was weakly recognized in both spermatogonial and immature Sertoli cells. These results indicate a direct involvement of AQP 1 in the regulation of fluid transport across the endothelial cell membranes of testicular microvessels. A role of the C-kit receptor protein is also substantiated by its strong expression in the maturing spermatogonia of the normal testes, but was minimally or not recognizable in undescended prepubertal testes.

Bench-to-bedside review: The role of the alveolar epithelium in the resolution of pulmonary edema in acute lung injury

Clearance of pulmonary edema fluid is accomplished by active ion transport, predominantly by the alveolar epithelium. Various ion pumps and channels on the surface of the alveolar epithelial cell generate an osmotic gradient across the epithelium, which in turn drives the movement of water out of the airspaces. Here, the mechanisms of alveolar ion and fluid clearance are reviewed. In addition, many factors that regulate the rate of edema clearance, such as catecholamines, steroids, cytokines, and growth factors, are discussed. Finally, we address the changes to the alveolar epithelium and its transport processes during acute lung injury (ALI). Since relevant clinical outcomes correlate with rates of edema clearance in ALI, therapies based on our understanding of the mechanisms and regulation of fluid transport may be developed.

Importance of type IV collagen, laminin, and heparan sulfate proteoglycan in the regulation of labyrinthine fluid in the rat cochlear duct.

The distribution of major components of the basement membrane, such as type IV collagen, laminin, and heparan sulfate proteoglycan (HSPG), was investigated in the rat cochlear duct. Immunofluorescence demonstrated that type IV collagen, laminin and HSPG were distributed along capillaries in the cochlear duct, including the stria vascularis, spiral ligament, spiral prominence and spiral limbus. Additionally, type IV collagen, laminin and HSPG were found to be distributed from the basement membrane of Reissner's membrane to that of the spiral prominence in a linear pattern. The scala media was surrounded by these basement membrane components, demarcating endolymph from perilymph, along epithelial cells except at the stria vascularis. These findings suggest that type IV collagen, laminin and HSPG create the anatomical separation between endolymph and perilymph, thus indicating that they may be involved in the regulation of fluid transport between the endolymph and perilymph.

Allatostatin-like-immunoreactive neurons of the tobacco hornworm, Manduca sexta, and isolation and identification of a new neuropeptide related to cockroach allatostatins.

The YXFGLamide C-terminus serves to define most members of a family of structurally related neuropeptides, the YXFGLamides. These peptides have been identified from the nervous system of various insects and include the allatostatins of cockroaches and crickets, the schistostatins of locusts, and the callatostatins of blowflies. The YXFGLamides have been shown to have various functions, including inhibition of juvenile hormone biosynthesis in cockroaches and crickets and inhibition of contraction of certain insect visceral muscles. We wanted to know if these peptides occur in Manduca sexta and what functions they might have. A new peptide, AKSYNFGLamide, was isolated and identified from M. sexta and has been named "lepidostatin-1"; this is the first YXFGLamide to be found in a lepidopteran, and there are indications that additional YXFGLamides occur in M. sexta. An antiserum to cockroach allatostatins (YXFGLamides) was shown to recognize lepidostatin-1 of M. sexta and was used to map YXFGLamide-immunoreactive neurons in larvae. Because immunoreactive interneurons were found to form an extensive neuropil, YXFGLamides probably function as neuromodulators in M. sexta. Neuroendocrine cells in the brain, abdominal ganglia, and their respective neurohemal organs were YXFGLamide immunoreactive and appear to release YXFGLamides as neurohormones. Immunoreactivity to YXFGLamides and M. sexta diuretic hormone were found to be colocalized and appear to be coreleased in these neuroendocrine cells, indicating that YXFGLamides may be involved in regulation of fluid transport. Innervation of the corpora allata by YXFGLamide-immunoreactive processes was very sparse, suggesting that this innervation does not play an important role in allatostasis. Many thoracic motor neurons were YXFGLamide immunoreactive, suggesting that YXFGLamides may have a myomodulatory or myotrophic function in larvae. However, this immunoreactivity disappeared early in metamorphosis and did not reappear in the adult. The YXFGLamide-immunoreactive neurons in the terminal abdominal ganglion were found to innervate the hindgut, indicating that YXFGLamides may be involved in the control of the rate of myogenic contractions of the larval hindgut.

Mechanisms of fluid losses in small‐bowel obstruction

Abstract: Small‐bowel obstruction (SBO) is a serious condition induced by hindrance of the propagation of intestinal contents. Mortality of 5% is still relatively high, but has decreased since the beginning of this century when it was around 60%. The condition is characterized by severe abdominal pain, accumulation of gas and fluids in the obstructed intestine and dehydration of the patient. The mechanisms responsible for the profuse fluid losses remain unclear. The aim of this thesis was to elucidate some aspects of the pathophysiology by using an experimental in vivo model in rats. In anaesthestized animals jejunum was ***string‐ligated approximately 10 cm distal of the ligament of Treitz. The laparotomy was closed and the animals allowed to recover. Next day the animals were reanaesthetized, tracheostomy was performed and the femoral artery and vein catheterized. Complete obstruction of the jejunum during 18 h resulting in fluid secretion which was subsequently measured using a gravimetric technique allowing continuous registration of intestinal net fluid transport (NFT). After a control period of 30 min with stable net fluid secretion drugs or saline were administered. Mean NFT of the following 2 h was compared to the mean NFT of the control period. In some animals Evans blue dye (20 mg · kg−1) was given as marker of blood vessel permeability. Some aspects of the study were investigated in human gastrointestinal tissue.Results show that a pronounced inflammation occurs in the obstructed bowel wall, as suggested by extravasation of plasma Evans blue‐albumin visualized by fluorescence microscopy and quantified by spectrophotometry. This was paralleled by net fluid secretion in the obstructed intestine. The importance of inflammation for the mechanisms of fluid losses was illustrated by the actions of the anti‐inflammatory agents, hydrocortisone and indomethacin, which reversed secretion into net fluid absorption. We proposed that the inflammation‐induced secretion of SBO was partly mediated through the enteric nervous system (ENS). This is supported by results showing that lidocaine, a local anaesthetic, with anti‐inflammatory properties, inhibited both inflammation and secretion, and anti‐inflammatory action of lidocaine was also shown in a human model where it reduced release of prostaglandins. The importance of ENS was further supported by results showing that the neurotransmitters vasoactive intestinal peptide (VIP) and somatostatin are involved in the regulation of fluid transport. VIP antiserum significantly reduced the net secretion, while the somatostatin analogue octreotide reversed net secretion into net absorption. Octreotide, which inhibits the release of many peptides including VIP, also reduced the Evans blue‐albumin extravasation. The secretion of SBO was also shown to be under influence of the sympathoadrenal system since clonidine, an α2‐adrenoceptor agonist, and propranolol, a β‐adrenoceptor antagonist, inhibited net fluid secretion. There were no significant effects on NFT after administration of α1‐ or β1‐adrenoceptor agonists or antagonists.In conclusion, the present work shows that obstruction of the small intestine will induce a pronounced inflammatory reaction in the bowel wall, most prominent in the external muscle layer. This inflammation probably triggers the profuse fluid losses by direct action on the enterocytes and by activation of enteric neurons. The anti‐inflammatory agents hydrocortisone, indomethacin, lidocaine and octreotide significantly reduced both the fluid losses and the increased Evans blue‐albumin extravasation. Neuropeptides of the ENS are also important, and neutralization of VIP using VIP antiserum or inhibiting the release of VIP by octreotide both reduced fluid losses. The fluid transport in obstructed gut seems also to be under tonic control of adrenergic neurons, as stimulation of α2‐adrenoceptors and blockade of β2‐adrenoceptors reduced fluid losses, while blockade of α2‐adrenoceptors and stimulation of β2‐adrenoceptors increased the net fluid secretion. Treatment of patients with small bowel obstruction with anti‐inflammatory drugs may prove beneficial

Fluid and electrolyte transport by cultured human airway epithelia.

An understanding of the fluid and electrolyte transport properties of any epithelium requires knowledge of the direction, rate, and regulation of fluid transport and the composition of the fluid. Although human airway epithelial likely play a key role in controlling the quantity and composition of the respiratory tract fluid, evidence for such a role is not available. To obtain such knowledge, we measured fluid and electrolyte transport by cultured human nasal epithelia. Under basal conditions we found that epithelia absorbed Na+ and fluid; both processes were inhibited by addition of amiloride to the mucosal surface. These data suggest that active Na+ absorption is responsible for fluid absorption. Interestingly, Na+ absorption was not accompanied by the net absorption of Cl-; some other anion accompanied Na+. The combination of cAMP agonists and mucosal amiloride stimulated the secretion of NaCl-rich fluid. But surprisingly, the response to cAMP agonists in the absence of amiloride showed substantial intersubject variability: cAMP stimulated fluid secretion across some epithelia, for others, cAMP stimulated fluid absorption. The explanation for the differences in response is uncertain, but we speculate that the magnitude of apical membrane Na+ conductance may modulate the direction of fluid transport in response to cAMP. We also found that airway epithelial secrete H+ and absorb K+ under basal conditions; both processes were inhibited by cAMP agonists. Because the H+/K(+)-ATPase inhibitor, SCH 28080, inhibited K+ absorption, an apical membrane H+/K(+)-ATPase may be at least partly responsible for K+ and H+ transport. However, H+/K+ exchange could not entirely account for the luminal acidification. The finding that cAMP agonists inhibited luminal acidification may be explained by the recent finding that cAMP increases apical HCO3- conductance. These results provide new insights into how the intact airway epithelium may modify the composition of the respiratory tract fluid.

The effect of secretin on sodium ion absorption by the isolated human gallbladder

Sodium ion (Na+) transport, a principal function of the gallbladder epithelium, was studied by measuring the flux of 22Na across isolated human gallbladder mucosa maintained in a modified 'Ussing' flux chamber. Tissue was obtained from cholecystectomy specimens in symptomatic patients with cholelithiasis. Out of 26 gallbladders studied, 13 had a net Na+ flux from mucosa to serosa which indicated active Na+ absorption. The hormone secretin, when added to the serosal fluid, reversed the direction of net flux in these gallbladders and caused a secretion of Na+ from serosa to mucosa. These results suggest that secretin may be involved in the physiological regulation of fluid transport in the human gallbladder, and also suggest a possible role for this hormone in gallbladder emptying.

Pump and leak in regulation of fluid transport in rabbit cornea.

Rabbit corneas were isolated, denuded of epithelium, and perfused on the anterior and posterior surfaces with Krebs Ringer-bicarbonate with additions of 50 microM H2O2, 125 microM BCNU, or 100 microM ouabain. The permeability of the corneal endothelium to labelled mannitol and inulin was determined by adding these compounds to the endothelial perfusate and measuring the rate of appearance of radioactivity in the anterior perfusate. Both H2O2 and BCNU increased the flux of mannitol and inulin across the endothelium in a time dependent manner, but ouabain had no effect. Additions of glucose with H2O2 or of GSH with BCNU prevented the observed changes in permeability. ATPase activities in the endothelia of intact, isolated corneas were also determined following incubation in the same media. The only observable effects of H2O2 and BCNU were slight reductions in the activity of Na+ + K+ ATPase. It is concluded that permeability changes, the leak, are more critical than active transport processes, the pump, in determining the rate and extent of swelling that results from exposure of the cornea to these agents.

Psychophysiology of conditioned emotional distrubances in humans.

ABSTRACTA discriminative avoidance conditioning technique was used to study urinary excretion of selected adrenal hormones in response to a stimulus which had acquired conditioned noxious properties through association with availability of punishment. A four day test procedure was employed: (1) to habituate subjects to the test environment; (2) obtain control data; (3) condition subjects; and (4) test reactions to the conditioned noxious stimulus. Urine samples were taken at two‐hour intervals preceding and following each of the four trials, and were analyzed for epinephrine, norepinephrine, total 17‐hydroxycorticosteroids, and other urinary constituents. These results were correlated with results obtained from monitoring of heart rate, skin resistance, blood pressure, and three measures of panel monitoring performance. Data analyses revealed significant changes from control levels during the test period for each of the principal measures described above and some specification of life systems interrelationships through correlation and factor analyses. Factors were identified which related to behavioral efficiency, psychological effort, fluid transport regulation, cardiovascular‐adrenal, and specific epinephrine and norepinephrine factors.