Altered Ion Transport Research Articles

Modification of biophysical properties of lung epithelial Na+ channels by dexamethasone

There is considerable interest in identifying the basic mechanisms by which dexamethasone alters ion transport across the adult alveolar epithelium. Herein, we incubated synchronized A549 cells, a human alveolar epithelial cell line, with dexamethasone (1 microM) for 24-48 h. When normalized to HPRT (a housekeeping gene), A549 beta- and gamma-subunit mRNA levels for the human amiloride-sensitive epithelial sodium channel (hENaC), assessed by RT-PCR, increased by 1.6- and 17-fold respectively, compared with control values (P < 0.05). These changes were abolished by actinomycin D, indicating transcriptional regulation. Western blotting studies revealed that dexamethasone also increased expression of beta- and gamma-hENaC protein levels. In contrast, alpha-hENaC mRNA increased by onefold (P > 0.05) and alpha-hENaC protein level was unchanged. Incubation of A549 cells with dexamethasone increased their whole cell amiloride-sensitive sodium currents twofold and decreased the K(0.5) for amiloride from 833 +/- 69 to 22 +/- 5.4 nM (mean +/- SE; P < 0.01). Single channel recordings in the cell-attached mode showed that dexamethasone treatment increased single channel open time and open probability threefold and decreased channel conductance from 8.63 +/- 0.036 to 4. 4 +/- 0.027 pS (mean +/- SE; P < 0.01). We concluded that dexamethasone modulates the amiloride-sensitive Na(+) channels by differentially regulating the expression of beta- and gamma-subunits at the mRNA and protein levels in the human A549 cell line, with little effect on alpha-hENaC subunit.

Identification of receptors for neuromedin U and its role in feeding.

Neuromedin U (NMU) is a neuropeptide with potent activity on smooth muscle which was isolated first from porcine spinal cord and later from other species. It is widely distributed in the gut and central nervous system. Peripheral activities of NMU include stimulation of smooth muscle, increase of blood pressure, alteration of ion transport in the gut, control of local blood flow and regulation of adrenocortical function. An NMU receptor has not been molecularly identified. Here we show that the previously described orphan G-protein-coupled receptor FM-3 (ref. 15) and a newly discovered one (FM-4) are cognate receptors for NMU. FM-3, designated NMU1R, is abundantly expressed in peripheral tissues whereas FM-4, designated NMU2R, is expressed in specific regions of the brain. NMU is expressed in the ventromedial hypothalamus in the rat brain, and its level is significantly reduced following fasting. Intracerebroventricular administration of NMU markedly suppresses food intake in rats. These findings provide a molecular basis for the biochemical activities of NMU and may indicate that NMU is involved in the central control of feeding.

Alterations of plasma calcium and intracellular and membrane calcium in erythrocytes of patients with pre-eclampsia.

Changes in plasma and intracellular calcium levels have been suggested in the pathogenesis of pre-eclampsia, however, membrane calcium content has not been studied so far. We compared intracellular and membrane calcium concentrations in erythrocytes of women with pre-eclampsia, healthy pregnant woman and controls to determine a possible alteration of membrane calcium in pre-eclampsia. Eighteen untreated, healthy pregnant woman and 16 pregnant nulliparous women with manifest pre-eclampsia were included, 25 healthy, age-matched woman served as controls. Atomic absorption spectroscopy was used for measurement of intracellular and membrane calcium content in erythrocytes and plasmalemmal preparations. Plasma Ca++ concentrations were significantly lower in pre-eclamptic women (1.96 +/- 0.15 mmol/l, P < 0.01, mean +/- s.e.m.) compared to healthy controls (2.43 +/- 0.14 mmol/l) or women with uncomplicated pregnancies (2.20 +/- 0.10 mmol/l). Intracellular Ca++ concentrations were not different between groups, however, membrane Ca++ content was significantly increased in the pre-eclamptic patients (1.23 +/- 0.36 micromol/g membrane protein, P < 0.01) compared to control subjects (0.83 +/- 0.16 micromol/g) and healthy pregnant women (0.77 +/- 0.13 micromol/g). Membrane calcium content is significantly increased in pre-eclamptic women despite low plasma Ca++ concentrations. This finding suggests an altered membrane ion transport and may be of importance for the pathogenesis of pre-eclampsia.

Perturbed bioelectrical properties of the mouse cecum following hepatectomy and starvation: the role of bacterial adherence.

Previous work in our laboratory has demonstrated that bacterial adherence alone to the intestinal epithelium, as occurs following catabolic stress, significantly perturbs the normal electrophysiology of the cecal mucosa. The aim of this study was to further characterize these effects in the mouse cecum following hepatectomy and short-term starvation, and to define the role of bacterial adherence in this process. Groups of mice underwent a surgical hepatectomy and were either fed or starved during the postoperative period. Groups of controls underwent sham operations and were either fed or starved postoperatively. Electrophysiologic studies in Ussing chambers at 48 hours were performed. Bacterial adherence to the mucosa was assessed by culture and histologic staining. To determine the role of bacteria in the altered electrophysiologic response, ciprofloxacin decontamination studies were performed. Only mice subjected to both hepatectomy and starvation developed bacterial adherence of sufficient magnitude (>10(5) cfu/gm) to alter mucosal electrophysiology (short-circuit current and basal potential difference). Ciprofloxacin decontamination completely abrogated this effect. Ion replacement studies suggested that active sodium transport was primarily responsible for the observed changes in mucosal electrophysiology. Bacterial-epithelial cell interactions may be responsible for altered mucosal ion transport observed following operative catabolic stress and short-term starvation.

Platelet Activating Factor Alters Ion Transport and Barrier Function in Intestinal Epithelial Cells

Platelet Activating Factor Alters Ion Transport and Barrier Function in Intestinal Epithelial Cells

Human colonic epithelial cells express galanin-1 receptors, which when activated cause Cl- secretion.

Galanin is a peptide hormone widely expressed in the central nervous system and gastrointestinal (GI) tract. Within the GI tract galanin is present in enteric nerve terminals where it is known to modulate intestinal motility by altering smooth muscle contraction. Recent studies also show that galanin can alter intestinal short-circuit current (Isc) but with differing results observed in rats, rabbits, guinea pigs, and pigs. In contrast, nothing is known about the ability of galanin to alter ion transport in human intestinal epithelial tissues. By RT-PCR, we determined that these tissues express only the galanin-1 receptor (Gal1-R) subtype. To evaluate Gal1-R pharmacology and physiology, we studied T84 cells. Gal1-R expressed by these cells bound galanin rapidly (half time 1-2 min) and with high affinity (inhibitor constant 0.7 +/- 0.2 nM). T84 cells were then studied in a modified Ussing chamber and alterations in Isc, a measure of all ion movement across the tissue, were determined. Maximal increases in Isc were observed in a concentration-dependent manner around 2 min after stimulation with peptide, with 1 microM galanin causing Isc to rise more than eightfold and return to baseline occurring within 10 min. The increase in galanin-induced Isc was shown by 125I efflux studies to be due to Cl- secretion, which occurred independently of alterations in cAMP and phospholipase C. Rather, Cl- secretion is mediated via a Ca2+-dependent, pertussis toxin-sensitive mechanism. These data suggest that galanin released by enteric nerves may act as a secretagogue in the human colon by activating Gal1-R.

AVP V1 receptor-mediated decrease in Cl- efflux and increase in dark cell number in choroid plexus epithelium.

The cerebrospinal fluid (CSF)-generating choroid plexus (CP) has many V1 binding sites for arginine vasopressin (AVP). AVP decreases CSF formation rate and choroidal blood flow, but little is known about how AVP alters ion transport across the blood-CSF barrier. Adult rat lateral ventricle CP was loaded with 36Cl-, exposed to AVP for 20 min, and then placed in isotope-free artificial CSF to measure release of 36Cl-. Effect of AVP at 10(-12) to 10(-7) M on the Cl- efflux rate coefficient (in s-1) was quantified. Maximal inhibition (by 20%) of Cl- extrusion at 10(-9) M AVP was prevented by the V1 receptor antagonist [beta-mercapto-beta, beta-cyclopentamethyleneproprionyl1,O-Me-Tyr2,Arg8]vasopressin. AVP also increased by more than twofold the number of dark and possibly dehydrated but otherwise morphologically normal choroid epithelial cells in adult CP. The V1 receptor antagonist prevented this AVP-induced increment in dark cell frequency. In infant rats (1 wk) with incomplete CSF secretory ability, 10(-9) M AVP altered neither Cl- efflux nor dark cell frequency. The ability of AVP to elicit functional and structural changes in adult, but not infant, CP epithelium is discussed in regard to ion transport, CSF secretion, intracranial pressure, and hydrocephalus.

Angiotensin II modulates respiratory and acid-base responses to prolonged hypoxia in conscious dogs

We tested the hypothesis that angiotensin II (ANG II) contributes to ventilatory and acid-base adaptations during 3-4 h of hypoxia (partial pressure of O2 in arterial blood approximately 43 Torr) in the conscious dog. Three protocols were carried out over 3-4 h in five dogs: 1) air control, 2) 12% O2 breathing, and 3) 12% O2 breathing with ANG II receptors blocked by infusion of saralasin (0. 5 microg . kg-1 . min-1). After 2 h of hypoxia, expired ventilation and alveolar ventilation progressively increased, and the partial pressure of CO2 in arterial blood and the difference between the arterial concentrations of strong cations and strong anions ([SID]) decreased. When the hypoxic chemoreceptor drive to breathe was abolished transiently for 30 s with 100% O2, the resultant central apneic time decreased between 0.5 and 2.5 h of hypoxia. All these adaptive responses to hypoxia were abolished by ANG II receptor block. Because plasma ANG II levels were lower during hypoxia and hypoxic release of arginine vasopressin from the pituitary into the plasma was prevented by ANG II receptor block, the brain renin-angiotensin system was likely involved. It is possible that ANG II mediates ventilatory and acid-base adaptive responses to prolonged hypoxia via alterations in ion transport to decrease [SID] in brain extracellular fluid rather than acting by a direct neural mechanism.

Alterations in Membrane Fatty Acid Unsaturation and Chain Length in Hypertension as Observed by 1H NMR Spectroscopy

Alterations in fatty acids of membrane phospholipids in essential hypertension may account for altered membrane ion transport, elasticity, and contractility properties of hypertensive tissues. To investigate the abnormalities in membrane fatty acids in essential hypertension, the degree of fatty acid unsaturation ([–CHCH–]/[–CH 3]), the average carbon chain length, ratio of glycerol to fatty acyl chains, ratio of phosphatidylcholine to fatty acyl chains, and the ratio of free and acylated cholesterol to fatty acyl chains in fatty acid fractions of membrane phospholipids of aorta, kidney, and heart were determined in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats by 1H nuclear magnetic resonance (NMR) spectroscopy. The degrees of fatty acid unsaturation in the aorta and the kidney membranes were significantly lower in SHR than in WKY rats (aorta, 0.53 ± 0.01 v 0.63 ± 0.01, n = 5, P = .01; kidney, 0.70 ± 0.01 v 0.84 ± 0.03, n = 10, P = .01). No significant difference could be detected in fatty acid unsaturation in heart membranes between these two strains. For aorta, kidney, and heart membranes, the average carbon chain lengths of fatty acid fractions of membrane phospholipids were significantly shorter for SHR than for WKY rats (aorta, 15.1 ± 0.2 v 18.3 ± 0.7, n = 5, P = .02; kidney, 14.5 ± 0.2 v 16.4 ± 0.4, n = 10, P = .01; heart, 17.3 ± 0.5 v 18.8 ± 0.6, n = 10, P = .05). The lower unsaturated fatty acid content in membrane phospholipids of the aorta and the kidney, with concomitant reduction in average chain length, may arise from increased oxidation of fatty acid double bonds in hypertensive tissues and may account, in part, for the increased aortic stiffness and abnormal kidney function associated with essential hypertension. Whether the lower unsaturated fatty acid content and decreased carbon chain length of phospholipid membranes in the aorta and the kidney are a cause or a consequence of the high blood pressure, however, remains unknown.

Study of the allergic alteration of ion transport in intestinal and tracheal epithelia of the guinea pig

Study of the allergic alteration of ion transport in intestinal and tracheal epithelia of the guinea pig

Mast cell mediated ion transport in intestine from patients with and without inflammatory bowel disease

Background—Mast cells have been shown to regulate intestinal ion transport in animal models and normal human colon but their physiological role in human intestinal inflammatory disorders is unknown.Aims—To examine mast...

Colonic Structural and Ion Transport Abnormalities in Suckling Rabbits Infected with Escherichia coli K12

BackgroundEscherichia coli K12 is a laboratory strain considered nonpathogenic. The purpose of this study was to examine the effect of E. coli K12 infection on colonic structure and function.MethodsSuckling rabbits were infected at 10 days of age with 6 × 109 CFU E. coli by intragastric inoculation and were examined 4 to 5 days later. Segments of ileum and proximal and distal colon were removed for light and electron microscopy, and NaCl transport was examined in vitro under short‐circuited conditions in Ussing chambers.ResultsInfection did not cause weight loss or diarrhea. Colonic mucosa was inflamed with infiltration by polymorpho‐nuclear neutrophils mainly in the lamina propria. The proximal and distal colon exhibited reduced Na+ absorption. The proximal colon also showed increased Cl‐ secretion; the ileum was unaffected.ConclusionsInfection with E. coli K12 disrupts the epithelium and alters ion transport in the colon, probably as a result of mucosal inflammation. The changes indicate that nonpathogenic E. coli have the potential to cause intestinal disease.

Colonic structural and ion transport abnormalities in suckling rabbits infected with Escherichia coli K12.

Escherichia coli K12 is a laboratory strain considered nonpathogenic. The purpose of this study was to examine the effect of E. coli K12 infection on colonic structure and function. Suckling rabbits were infected at 10 days of age with 6 x 10(9) CFU E. coli by intragastric inoculation and were examined 4 to 5 days later. Segments of ileum and proximal and distal colon were removed for light and electron microscopy, and NaCl transport was examined in vitro under short-circuited conditions in Ussing chambers. Infection did not cause weight loss or diarrhea. Colonic mucosa was inflamed with infiltration by polymorphonuclear neutrophils mainly in the lamina propria. The proximal and distal colon exhibited reduced Na+ absorption. The proximal colon also showed increased Cl- secretion; the ileum was unaffected. Infection with E. coli K12 disrupts the epithelium and alters ion transport in the colon, probably as a result of mucosal inflammation. The changes indicate that nonpathogenic E. coli have the potential to cause intestinal disease.

Plasma triglycerides and red cell ion transport alterations in genetically hypertensive rats.

Ion transport abnormalities in essential hypertension are often associated with concomitant changes of lipid metabolism, but this information is missing in rats with genetic hypertension. We therefore studied the alterations of red cell Na+ and K+ transport and their relationship to blood pressure and plasma lipids (cholesterol and triglycerides) in Prague hereditary hypertriglyceridemic (HTG) rats, Lyon hypertensive (LH) rats, and HTG x Lewis F2 hybrids. In both hypertensive models and F2 hybrids, red cell Na+ content (Na+(i)) was positively related to plasma triglycerides but not to plasma cholesterol levels. Na+(i) elevation was more pronounced in HTG than in LH rats, probably due to higher plasma triglycerides in the former strain. The two hypertensive strains differed in bumetanide-sensitive Na+ transport, which was augmented in HTG rats with low plasma cholesterol but suppressed in LH rats characterized by high cholesterol levels. In the two genetic models, there was a positive association of blood pressure with Na+ leak, and this was also confirmed by the cosegregation of these parameters in F2 hybrids. We conclude that the enhancement of Na+ leak represents the major ion transport abnormality in rats with genetic hypertension. The alterations in plasma lipids are important determinants of abnormal red cell ion transport in hypertensive models studied. Although the detailed mechanism of their participation in ion transport regulation is still not completely understood, triglyceride-dependent changes in membrane microviscosity seem to be responsible for the modulation of particular ion transport pathways.

Relationship of red blood cell ion transport alterations and serum lipid abnormalities in Lyon genetically hypertensive rats

Alterations of Na+ and K+ transport in erythrocytes of hypertensive humans or animals are often associated with abnormal lipid metabolism. The aim of the present study was to investigate red blood cell ion transport in Lyon inbred strains selected from Sprague-Dawley rats for different blood pressure levels. Lyon strains are characterized by important metabolic changes, including plasma lipid abnormalities. Serum triglycerides, cholesterol, and uric acid as well as red blood cell Na+ and K+ (Rb+) transport mediated by Na(+)-K+ pump or Na(+)-K+ cotransport and cation leaks were studied in hypertensive (LH), normotensive (LN), and low blood pressure (LL) Lyon rats aged 12 weeks. Increased erythrocyte Na+ content (Nai+) and higher levels of serum triglycerides, cholesterol, and uric acid were demonstrated in LH rats compared with LN animals. Nevertheless, at this age serum triglycerides and erythrocyte Nai+ of LL rats were even higher than those of LH animals. There were no significant differences between Lyon strains in either Na(+)-K+ pump activity or bumetanide-resistant (BR) cation leaks. The activity of bumetanide-sensitive (BS) Na(+)-K+ cotransport mediating inward Na+ movement was highest in LL rats and lowest in LH animals. In Lyon rats, Nai+ was positively related to serum triglycerides, whereas blood pressure correlated positively with BR Na+ leak and negatively with BS net Na+ uptake. A similar association of erythrocyte Nai+ with serum triglycerides was also observed in Prague hereditary hypertriglyceridemic rats (HTG) that were selected from Wistar rats for high plasma triglycerides. The major difference of the two forms of genetic hypertension associated with abnormal lipid metabolism was in BS net Na+ uptake, which was enhanced in HTG but reduced in LH rats. This was probably due to differences in plasma cholesterol, which was elevated in LH but not in HTG animals. Our study in Lyon rats confirmed the positive association of blood pressure with Na+ leak as a characteristic feature of genetic hypertension.

The effect of brefeldin A on terbutaline-induced sodium absorption in fetal rat distal lung epithelium.

We studied the effect of brefeldin A, which inhibits the intracellular trafficking of membrane proteins from the cytosolic pool to the cell surface, on terbutaline (a beta2-specific adrenergic agonist)-induced alterations in ion transport by primary monolayer cultures of fetal rat distal lung epithelium. The amiloride-sensitive short circuit current (Isc) increased 2.5-fold 50 min after application of terbutaline (10 microM) from basolateral side; this response was abolished by pretreatment with brefeldin A (1 microg/ml). Brefeldin A did not suppress the Na+/K+ pump capacity. Single channel patch clamp experiments demonstrated that terbutaline increased the density of amiloride-sensitive Na+-permeable nonselective cation channels on the apical cell membrane and this action was blocked by brefeldin A. These observations suggest that beta2-specific adrenergic agonists promote the trafficking of amiloride sensitive Na+-permeable nonselective cation channels to the apical cell surface.

Reactive oxygen metabolites in piglet cryptosporidiosis.

Piglet cryptosporidiosis is characterized by intestinal villous damage and malabsorption, and by reduced NaCl absorption in response to prostaglandins (PGs), which act directly on the epithelium and indirectly through enteric nerves. We hypothesized that phagocyte-derived reactive oxygen metabolite (ROM) production contributed to PG synthesis and altered transport in inflamed ileum. Ileal mucosa from control and infected piglets was analyzed for villous height, PGE2, catalase (an endogenous antioxidant), and malondialdehyde (MDA, a by-product of lipid peroxidation) from d 2-8 after infection. The response of control ileal mucosa to exogenous ROM and infected mucosa to antioxidant treatment was also studied in tissues mounted in Ussing chambers. Increased levels of MDA on d 2 preceded increased PGE2 on d 3-4, which correlated with the acute diarrheal phase; however the most severe villous atrophy (d 8) correlated with the highest levels of catalase and MDA but low levels of PGE2. Control mucosa responded to H2O2 with indomethacin- and tetrodotoxin-sensitive transient increases in short circuit current (Isc), which were accompanied by increased tissue production of 6-keto-PGF1a, the stable metabolite of PGI2; however, no increased PGE2 production was detectable. A stable analog of PGI2, carbacyclin, mimicked the transient Isc response to H2O2; however, several antioxidants failed to alter the abnormal Isc of infected tissue. These results suggest that there is evidence of increased ROM production in cryptosporidial infection and that intestinal PG synthesis and inhibited NaCl absorption may be mediated partially by ROM in this model. Additional, cooperative factors, such as PGE2 production, however, are likely needed to induce the alterations in ion transport seen in this infection.

Sequential changes in biliary lipids and gallbladder ion transport during gallstone formation.

This study sought to correlate gallbladder (GB) Na+ and Cl-) fluxes with biliary lipid composition during the various stages of gallstone (GS) formation. GS formation is associated with altered GB ion transport and increased biliary lipid and Ca2+ concentrations. Nonetheless, the longitudinal relationship between ion transport and biliary lipid changes during GS formation has not been defined. Prairie dogs were fed standard (n = 18) or 1.2% cholesterol-enriched (n = 30) diets for 4 to 21 days. Hepatic and GB bile were analyzed for lipids and Ca2+. Animals were designated either Pre-Crystal, Crystal, or GS based on absence or presence of crystals or GS, respectively. GBs were mounted in Ussing chambers, electrophysiologic parameters were recorded, and unidirectional Na+ and Cl- fluxes measured. Short-circuit current and potential difference were similar during Pre-Crystal and Crystal stages but significantly reduced during GS stage compared to controls and Pre-Crystals. Transepithelial resistance was similar in all groups. Net Na+ absorption was increased during Pre-Crystal but decreased during GS stage due to increased mucosa-to-serosa and serosa-to-mucosa flux, respectively. Increased serosa-to-mucosa flux of both Na+ and Cl- characterized the Crystal stage. Biliary lipids and Ca2+ increased progressively during various stages of GS formation and correlated positively with unidirectional fluxes of Na+ and Cl-. GB epithelial ion transport changes sequentially during GS formation, with the early Pre-Crystal stage characterized by increased Na+ absorption, and the later Crystal stage accompanied by prosecretory stimuli on Na+ and Cl- fluxes, which may be due to elevated GB bile Ca2+ and total bile acids.

Urea inhibits Na-K-2Cl cotransport in medullary thick ascending limb cells.

We examined the effect of physiological concentrations of urea (100-500 mM) on Na-K-2Cl cotransport in cultured cells from mouse medullary thick ascending limb (mTAL). Urea acutely inhibited bumetanide-sensitive K influx in mTAL cells in a concentration-dependent fashion, with a statistically significant inhibition (19%) at 100 mM and 86% inhibition at 500 mM. The effect of urea was entirely reversible and was blocked by prior treatment with okadaic acid, a phosphatase inhibitor, suggesting that urea exerts its action upstream of the phosphorylation-dephosphorylation step. Cell volume was unchanged in the presence of 500 mM urea. The number of [3H]bumetanide binding sites, a measure of the number of functioning cotransporter sites, was decreased in the presence of urea, and the decrease in bumetanide binding was proportional to the decrease in bumetanide-sensitive K influx. Urea also stimulated the Ba-sensitive swelling-activated K efflux from mTAL cells. Thus urea, in concentrations that prevail in the renal medulla, alters ion transport in mTAL cells.

Failure of thapsigargin to alter ion transport in human sweat gland epithelia while intracellular Ca2+ concentration is raised

Failure of thapsigargin to alter ion transport in human sweat gland epithelia while intracellular Ca2+ concentration is raised