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Lysyl oxidase like 2 (LOXL2) is associated with poor prognosis in idiopathic pulmonary disease (IPF) and cancer. We developed an Enzyme-linked immunosorbent assay (ELISA) targeting the LOXL2 neo-epitope generated through the release of the signal peptide during LOXL2 maturation.

ABSTRACTThe matricellular protein SPARC (secreted proteome acidic and rich in cysteine) is known to bind collagens and regulate fibrillogenesis. Cleavage of SPARC at a single peptide bond, increases the affinity for collagens up to 20-fold. To investigate if this specific cleavage has pathological relevance in fibrotic disorders, we developed a competitive ELISA targeting the generated neo-epitope on the released fragment and quantified it in serum from patients with lung cancer, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD) and healthy subjects. Furthermore, the ability of SPARC to protect fibrillar collagens from proteolytic degradation was investigated in vitro, potentially adding a new collagen chaperone function to SPARC. The fragment was significantly elevated in lung cancer patients when compared to healthy subjects measured in a discovery cohort (p = 0.0005) and a validation cohort (p < 0.0001). No significant difference was observed for IPF and COPD patients compared to healthy subjects. When recombinant SPARC was incubated with type I or type III collagen and matrix metalloproteinase-9, collagen degradation was completely inhibited. Together, these data suggest that cleavage of SPARC at a specific site, which modulates collagen binding, is a physiological mechanism increased during pathogenesis of lung cancer. Furthermore, inhibition of fibrillar collagen degradation by SPARC adds a new chaperone function to SPARC which may play additional roles in the contribution to increased collagen deposition leading to a pro-fibrotic and tumorigenic environment.

Cl- transport was studied in human nasal epithelium, a predominantly Na+-absorbing proximal airway epithelium. Intracellular Cl- activity (aClc) and the electrical potentials across the apical (Va) and basolateral (Vb) membranes were measured with double-barreled, Cl- -selective microelectrodes to characterize the driving forces for Cl- flow across each membrane. Under control conditions (bilateral Krebs-bicarbonate Ringer), Va was -26.1 +/- 1.2 mV, Vb was -36.2 +/- 1.2 mV, and aCL(c) was 42.7 +/- 2.0 mM (n = 34), indicating that Cl- is near electrochemical equilibrium across the apical membrane but significantly above equilibrium across the basolateral membrane. Reduction of luminal [Cl-] from 120 to 3 mM reduced aClc from 42.7 +/- 4.0 to 27.0 +/- 3.5 mM, depolarized Va, and increased fractional apical membrane resistance (fRa) and transepithelial resistance (Rt). Serosal bumetanide reduced aClc by 10 mM without affecting electrical parameters. Reduction of serosal [Cl-] from 120 to 3 mM resulted in a rapid decrease in Vb, a decrease in fRa and an increase in Rt. Also, serosal [Cl-] reduction led to a slow decrease in aClc rom 45.5 +/- 2.5 to 31.1 +/- 4.2 mM) that could be inhibited by bumetanide. The data are consistent with the following conclusions: 1) Cl- is transported across the apical membrane through a conductive pathway; and 2) Cl- is translocated across the basolateral membrane by an electrically silent bumetanide-sensitive cotransport system and by a minor conductive path.

A method for determination of shunt resistance (Rs) and absolute conductive ion permeabilities of the apical membrane in epithelia from steady-state data is described. The method assumes that the currents are satisfactorily described by the Goldman-Hodgkin-Katz regime. Its application requires measurements of standard transepithelial electrophysiological parameters and of one or more intracellular ion activities. It is applicable under both open- and short-circuit conditions. The method was tested in an electrophysiological analysis of cultured normal and cystic fibrosis (CF) human nasal epithelium. In 15 normal and 10 CF preparations with mean transepithelial resistances of 338 and 427 omega.cm2, Rs was 412 and 623 omega.cm2, respectively. The Rs values determined with the present method were strongly correlated (r = 0.94) with those obtained with another method available in the electrophysiological literature but were as a mean 20% lower. Amiloride increased Rs by 25% in CF and by 8% in normal preparations. In normal preparations, the apical Cl permeability (PCla) was 3.6 x 10(-6) cm/s, and the apical Na permeability (PNaa) was 1.6 x 10(-6) cm/s. In CF preparations, PCla was reduced to a maximum of 2.3 x 10(-7) cm/s, whereas PNaa was increased to 6.2 x 10(-6) cm/s. The apical membrane electromotive force was -1 mV in normal and 43 mV in CF preparations. It is concluded that the method can be used to calculate Rs, apical membrane ion permeabilities, and electromotive forces from steady-state electrophysiological data.

Cultured human nasal epithelia derived from cystic fibrosis (CF) patients were studied with double-barreled, Cl- -selective microelectrodes to measure membrane potentials and intracellular Cl- activity (aClc). The aClc of CF cultures was 46.5 +/- 2.5 mM (n = 28), a value not significantly different from aClc of normal human nasal cells. Reduction of the luminal [Cl-] from 120 to 3 mM failed to reveal any apical Cl- permeability (conductive or nonconductive) in CF cultures. Bumetanide (10(-4) M, serosal) led to a 10 mM decrease in aClc without affecting the electrical parameters of the cells. Reduction of serosal [Cl-] led to a marked decrease in aClc (from 58.0 +/- 6.7 to 26.8 +/- 2.9 mM) that could partly be blocked by bumetanide. Reduction of serosal [Cl-] led to a rapid depolarization (5.4 +/- 0.7 mV) of the basolateral membrane potential (Vb), a decrease of the fractional apical membrane resistance (0.03 +/- 0.01), and an increase (34 +/- omega.cm2) in the transepithelial resistance (Rt). We conclude that 1) the apical membrane of CF airway epithelia is impermeable to Cl-, and 2) Cl- transport across the basolateral membrane occurs mainly through a bumetanide-inhibitable cotransport system but also through a Cl- conductance, neither of which appears to be affected by CF.

Cystic fibrosis (CF) airway epithelia express a defect in adenosine 3',5'-cyclic monophosphate (cAMP)-dependent regulation of apical membrane Cl- channels. Recent patch-clamp studies have raised the possibility that Ca2+ -dependent mechanisms for the activation of Cl- secretion may be preserved in CF airway epithelia. To determine 1) whether intact normal (N1) and CF airway epithelia exhibit a Ca2+ -dependent mechanism for activation of Cl- secretion and 2) whether Ca2+ -dependent mechanism for activation of Cl- secretion and 2) whether Ca2+ -dependent mechanisms initiate Cl- secretion via activation of an apical membrane Cl- conductance (GCl-), nasal epithelia from N1 and CF subjects were cultured on collagen membranes, and responses to isoproterenol or Ca2- ionophores [A23187 10(-6) M; ionomycin (10(-5)M)] were measured with transepithelial and intracellular techniques. Isoproterenol induced activation of an apical membrane GCl- in N1 cultures but was ineffective in CF. In contrast, in both N1 and CF amiloride-pretreated cultures, A23187 induced an increase in the equivalent short-circuit current that was associated with an activation of an apical membrane Gc1- and was bumetanide inhibitable. A23187 addition during superfusion of the lumen with a low Cl- (3 mM) solution reduced intracellular Cl- activity of CF cells. A Ca2+ ionophore of different selectivity properties, ionomycin, was also an effective Cl- secretagogue in both N1 and CF cultures. We conclude that 1) the A23187 induced Cl- secretion via activation of an apical GCl- in N1 human nasal epithelium, and 2) in contrast to an isoproterenol-dependent path, a Ca2+ -dependent path for GCl- activation is preserved in CF epithelia.

1. The control by intracellular cyclic AMP and beta‐adrenergic stimulation of chloride conductance was studied in toad skin epithelium mounted in a chamber on the stage of an upright microscope. Impalement of identified principal cells from the serosal side with single‐barrelled conventional or double‐barrelled Cl(‐)‐sensitive microelectrodes was performed at x500 magnification. For blocking the active sodium current 50 microM‐amiloride was present in the mucosal bath. 2. When clamped at transepithelial potential difference V = 0 mV, the preparations generated clamping currents of 0.9 +/‐ 1 microA/cm2 (mean +/‐ S.E.M.; number of observations n = 55). The intracellular potential of principal cells (Vb) was ‐96 +/‐ 2 mV with a fractional resistance of the basolateral membrane (fRb) of 0.016 +/‐ 0.003 (n = 54), and an intracellular Cl‐ activity of 40 +/‐ 2 mM (n = 24). 3. At V = 0 mV, serosal application of a cyclic AMP analogue, dibutyryl cyclic AMP (500 microM) or a beta‐adrenergic agonist, isoprenaline (5 microM) resulted in a sixfold increase in transepithelial Cl‐ conductance identified by standard 36Cl‐ tracer technique. 4. The clamping current at V = 0 mV was unaffected by cyclic AMP (short‐circuit current Isc = 0.1 +/‐ 0.3 microA/cm2, n = 16) indicating that subepidermal Cl(‐)‐secreting glands are not functioning in our preparations obtained by collagenase treatment. 5. Cyclic AMP‐ or isoprenaline‐induced chloride conductance (Gcl) activation (V = 0 mV) was not reflected in membrane potential and intracellular Cl‐ activity in principal cells. Intracellular chloride activity was constant at approximately 40 mM at membrane potentials between ‐90 and ‐100 mV. Therefore, it can be concluded that the principal cells are not contributing to activated Cl‐ currents. 6. At V = ‐100 mV where the voltage‐dependent chloride conductance of mitochondria‐rich (MR) cells was already fully activated, GCl was unaffected by cyclic AMP or isoprenaline. The major effect of these treatments was a rightward displacement of the MR cell‐generated GCl‐V relationship along the V axis. 7. Our results indicate that the beta‐adrenergically controlled cyclic AMP‐mediated chloride conductance is localized to the mitochondria‐rich cells.

Summary The potential dependence of unidirectional36Cl fluxes through toad skin revealed activation of a conductive pathway in the physiological region of transepithelial potentials. Activation of the conductance was dependent on the presence of Cl- or Br- in the external bathing solution, but was independent of whether the external bath was NaCl-Ringer's, NaCl-Ringer's with amiloride, KCl-Ringer's or choline Cl-Ringer's To partition the routes of the conductive Cl- ion flow, we measured in the isolated epithelium with double-barrelled microelectrodes apical membrane potentialVa, and intracellular Cl- activity,aClc, of the principal cells indentified by differential interference contrast microscopy. Under short-circuit conditionsIsc=27.0±2.0 µA/cm2, with NaCl-Ringer's bathing both surfaces,Va was -67.9±3.8mV (mean ±se,n=24, six preparations) andaClc was 18.0±0.9mM in skins from animals adapted to distilled water. BothVa andaCla were found to be positively correlated withIsc (r=0.66 andr=0.70, respectively). In eight epithelia from animals adapted to dry milieu/tap waterVa andaClc were measured with KCl Ringer's on the outside during activation and deactivation of the transepithelial Cl- conductance (GCl) by voltage clamping the transepithelial potential (V) at 40 mV (mucosa positive) and -100 mV. AtV=40 mV; i.e. whenGCl was deactivated,Va was -70.1±5.0 mV (n=15, eight preparations) andaClc was 40.0±3.8mm. The fractional apical membrane resistance (fRa) was 0.69±0.03. Clamping toV=-100 mV led to an instantaneous change ofVa to 31.3±5.6 mV (cell interior positive with respect to the mucosal bath), whereas neitheraClc norfRa changed significantly within a 2 to 5-min period during whichGCl increased by 1.19±0.10 mS/cm2. WhenV was stepped back to 40 mV,Va instantaneously shifted to -67.8±3.9 mV whileaClc andfRa remained constant during deactivation ofGCl. Similar results were obtained in epithelia impaled from the serosal side. In 12 skins from animals adapted to either tap water or distilled water the density of mitochondria-rich (DMRC) cells was estimated and correlated with the Cl current (ICl though the fully activated (V=-100mV) Cl- conductance). A highly significant correlation was revealed (r=-0.96) with a slope of -2.6 nA/m.r. (mitochondria-rich cell and an I-axis intercept not significantly different from zero. In summary, the voltage-dependent Cl currents were not reflected infRa andaCla of the principal cells but showed a correlation with the m.r. cell density. We conclude that the pricipal cells do not contribute significantly to the voltage-dependent Cl conductance.

A new species of naked, multinucleated amoebae, Chaos zoochlorellae sp. nov., belonging to the family Amoebidae, is described. Observations were made on individuals collected repeatedly from a small fresh water pond. Limnological parameters in the pond and the various attempts to establish a culture are described. The new species is a medium-size amoeba with both polypodial and monopodial locomotive forms, comparatively few nuclei and zoochlorellae as a prominent cytoplasmic inclusion.

1. Active Cl‐ currents were studied in short‐circuited toad skin epithelium in which the passive voltage‐activated Cl‐ current is zero. Under visual control double‐barrelled microelectrodes were used for impaling principal cells from the serosal side, or for measuring the pH profile in the solution bathing the apical border. 2. The net inward (active) 36Cl‐ flux of 27 +/‐ 8 pmol s‐1 cm‐2 (16) (mean +/‐ S.E.M (number of observation)) was abolished by 2 mM‐CN‐ (6.3 +/‐ 3.5 pmol s‐1 cm‐2 (8)). The active flux was maintained in the absence of active Na+ transport when the latter was eliminated by either 100 microM‐mucosal amiloride, replacement of mucosal Na+ with K+, or by 3 mM‐serosal ouabain. 3. In Ringer solution buffered by 24 mM‐HCO3‐ ‐5% CO2 mucosal amiloride reversed the short circuit current (ISC). The outward ISC was maintained when gluconate replaced mucosal Cl‐, and it was reversibly reduced in CO2‐free 5 mM‐Tris‐buffered Ringer solution (pH = 7.40) or by the proton pump inhibitor oligomycin. These observations indicate that the source of the outward ISC is an apical proton pump. 4. Amiloride caused principal cells to hyperpolarize from a basolateral membrane potential, Vb, of ‐73 +/‐ 3 (22) to ‐93 +/‐ 1 mV (26), and superfusion with CO2‐free Tris‐buffered Ringer solution induced a further hyperpolarization (Vb = ‐101 +/‐ 1 mV (26)) which could be blocked by Ba2+. The CO2‐sensitive current changes were null at Vb = EK (potassium reversal potential, ‐106 +/‐ 2 mV (55)) implying that they are carried by K+ channels in the basolateral membrane. Such a response cannot account for the inhibition of the outward ISC which by default seems to be located to mitochondria‐rich (MR) cells. 5. In the absence of mucosal Cl‐ a pH gradient was built up above MR cells with pH = 7.02 +/‐ 0.04 (42) and pH increasing to 7.37 +/‐ 0.02 (10) above principal cells (pH = 7.40 in bulk solution buffered by 0.1 mM‐Tris). This observation localizes a proton pump to the apical membrane of MR cells. Using the integrated diffusion equation it was shown that the measured external pH gradient would account within an order of magnitude for measured currents. 6. Standing gradients of protons were eliminated in the presence of mucosal Cl‐ suggesting that active uptake of Cl‐ is associated with the exit of base equivalents across the apical membrane of MR cells.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Intracellular pH (pHi) of cultured human airway epithelial cells from normal and cystic fibrosis (CF) subjects were measured with double‐barrelled pH‐sensitive liquid exchanger microelectrodes. The cells, which were grown to confluence on a permeable collagen matrix support, were mounted in a modified miniature Ussing chamber. All studies were conducted under open circuit conditions. Values are given as means +/‐ S.E.M. and n refers to the number of preparations. 2. Normal preparations (n = 15) were characterized by a transepithelial potential difference (Vt) of ‐18 +/‐ 2 mV, an apical membrane potential (Va) of ‐19 +/‐ 2 mV, a basolateral membrane potential (Vb) of ‐37 +/‐ 2 mV, a transepithelial resistance (Rt) of 253 +/‐ 15 omega cm2, a fractional apical membrane resistance (fRa) of 0.40 +/‐ 0.04 and an equivalent short circuit current (Ieq) of ‐73 +/‐ 7 microA cm‐2. 3. CF preparations (n = 13) were characterized by a Vt of ‐46 +/‐ 7 mV, a Va of 3 +/‐ 5 mV, a Vb of ‐43 +/‐ 3 mV, Rt of 373 +/‐ 47 omega cm2, fRa of 0.44 +/‐ 0.04 and an Ieq of ‐130 +/‐ 16 microA cm‐2. All parameters except Vb and fRa were significantly different (P < 0.025) from those of normal preparations. 4. Despite large differences in electrochemical driving force for proton flow across the apical cell membranes between normal and CF preparations (‐4 +/‐ 3 mV and 20 +/‐ 7 mV, respectively), pHi was similar (7.15 +/‐ 0.02 and 7.11 +/‐ 0.05, respectively). The driving force across the basolateral membrane was similar in normal and CF preparations (22 +/‐ 3 and 26 +/‐ 3 mV, respectively). 5. Intracellular alkalinization achieved by removal of CO2 from the luminal Ringer solution or by luminal ammonium prepulse led to stimulation of Ieq in both normal (from ‐58 to ‐70 microA cm‐2, n = 4; P < 0.05) and CF (from ‐144 to ‐163 microA cm‐2, n = 4; P < 0.005) preparations. The increase in Ieq was associated with a reduction of Rt, increase in fRa, and hyperpolarization of Vb. All changes in bioelectric properties in response to intracellular alkalinization were fully reversible. 6. Intracellular acidification achieved by serosal ammonium prepulse led to marked reductions of Ieq in both normal (from ‐95 to ‐31 microA cm‐2, n = 6; P < 0.05) and CF (from ‐111 to ‐67 microA cm‐2, n = 7; P < 0.005) preparations.(ABSTRACT TRUNCATED AT 400 WORDS)

Hexeberg S, Willumsen N, Berge RK. Docosahexaenoic acid induces lipid accumulation in myocardial cells of rats. Scand J Clin Lab Invest 1994; 54: 665-71.The aim of the study was to explore whether treatment with highly purified docosahexaenoic acid (DHA) over a short period affects the amount of lipid droplets in myocardial cells of rats, and whether heart peroxisomal enzyme activity is changed. Fifteen rats were fed a standard diet for 10 days and 15 rats were fed a cholesterol diet (2% of cholesterol) for 10 days. In each experiment six rats served as control, and three rats in each treatment group were given one of the following treatments by gastric intubation: DHA at 500, 1000, or 1500mg day-1 kg-1 body weight. The fractional volume of lipid droplets in myocardial cells was calculated by morphometric methods. The heart triglycerides and the volume fraction of lipid droplets in the myocardium were greater in the standard diet rats treated with DHA compared with controls. There was no such increase caused by DHA treatment in the cholesterol diet rats. The heart fatty acyl-CoA oxidase tended to increase with DHA treatment in both standard and cholesterol diet rats, but this was significantly increased only after treatment with DHA 1500mg day-1 kg-1 in the cholesterol diet rats. We conclude that treatment with highly purified DHA for 10 days results in cardiac lipidosis, assessed both by biochemical and morphological methods in standard diet rats, whereas DHA treatment has no additive effect on lipid accumulation in cholesterol fed rats.

A large, discoid amoeba isolated from moss in an English wood was identified as belonging to the same species as an organism described from fresh water in Germany under the name Hyalodiscus placopus. Notable characters were the many narrow subpseudopodia by which it makes contact with the substratum during locomotion and a very flexible test which covers only the dorsal side in locomotion. This test or cuticle was found by electron-microscopy to be fibrous and spongiose in structure and by cytochemical tests to be at least partly polysaccharide in composition. On the basis of comparison with an organism described by Pussard. Senaud and Pons (1977), particularly the cuticle, this isolate is classified as Gocevia placopus (Hülsmann 1974), a member of the family Cochliopodiidae and the order Arcellinida. The nature of the cuticle of Gocevia now seems definitely established, although questions remain about some other characters of the genus. The status of the genus Hyalodiscus is still uncertain. Organisms of two different species from a pond in Denmark showed similarities to G. placopus suggesting that they belong to the same genus, although the presence of a cuticle is not certain.

The effect of docosahexaenoic acid (DHA) on mitochondrial and peroxisomal fatty acid oxidation and on key enzymes in triglyceride metabolism was investigated in the liver of rats fed a standard diet, a cholesterol diet, and a pelleted chow diet. Unexpectedly, in all three rat models repeated administration of highly purified DHA (92% pure) at different doses and times, at a dose of 1000 mg/day per kg body weight, resulted in no significant decrease of hepatic and plasma concentration of triglycerides. The serum concentrations of cholesterol and phospholipids showed an increase in a time-dependent manner in rats fed the pelleted chow diet. The hepatic concentration of cholesterol was increased in rats fed the cholesterol diet and pelleted chow diet after administration of DHA compared to palmitic acid. In all rat models, treatment with DHA tended to increase the peroxisomal beta-oxidation. This was accompanied with a significant increase (1.5-fold) of fatty acyl-CoA oxidase activity. The mitochondrial fatty acid oxidation system and carnitine palmitoyl-transferase activity, however, were almost unchanged. Moreover, palmitoyl-CoA synthetase activity was increased, whereas the palmitoyl-CoA hydrolase activity was decreased. Neither microsomal phosphatidate phosphohydrolase activity nor cytosolic phosphatidate phosphohydrolase activity was affected by DHA feeding in the three rat models. Acyl-CoA:1,2-diacylglycerol acyltransferase activity was also unaffected. In contrast to docosahexanoic acid feeding, eicosapentaenoic acid (EPA) administration possessed a hypotriglyceridemic effect and resulted in an increase of mitochondrial and peroxisomal oxidation of fatty acids. Carnitine palmitoyltransferase activity was also stimulated. Phosphatidate phosphohydrolase activity was unaffected whereas diacylglycerol acyltransferase activity was increased by EPA treatment compared with palmitic acid feeding. The results indicate that docosahexaenoic acid, in contrast to eicosapentaenoic acid, does not inhibit the synthesis and secretion of triglycerides in the liver. In addition, the results emphasize the importance that stimulation of peroxisomal beta-oxidation by these n-3 fatty acids is not sufficient to decrease the serum levels of triglycerides. In addition, increased mitochondrial beta-oxidation of fatty acids and thereby decreased availability of nonesterified fatty acids may be a mechanism by which EPA inhibits triglyceride, and subsequently very low density lipoprotein-triglyceride, production. Whether DHA and EPA possess different metabolic properties should be considered.

Human airway epithelia are predominantly Na(+)-absorbing epithelia. To investigate the mechanisms for Na+ absorption across airway epithelia, the driving forces and paths for Na+ translocation across each membrane were examined with double-barreled Na(+)-selective microelectrodes in cultured human nasal epithelium (HNE). Under control conditions, intracellular Na+ activity (acNa) was 23 +/- 1 mM (n = 44 preparations, 393 impalements). Amiloride (10(-4) M) hyperpolarized the apical membrane and increased the fractional apical membrane resistance but did not affect acNa. Exposure to Na(+)-free luminal solution induced bioelectric responses similar to amiloride but also reduced acNa to 8 +/- 1 mM. Reduction of luminal Na+ concentration ([Na+]) in the presence of amiloride also reduced acNa without further changes in bioelectric parameters. Reduction of serosal [Na+] decreased aNac, a response blocked by bumetanide (10(-4) M). Ouabain (10(-4) M, serosal) led to a reduction in equivalent short-circuit current (Ieq) and increase in acNa. We conclude that 1) acNa is higher in HNE than in most mammalian epithelial cells, 2) the apical membrane expresses a conductive Na+ path, and 3) the basolateral membrane transports Na+ via the Na(+)-K(+)-adenosinetriphosphatase and a Na(+)-K(+)-2Cl- cotransport system.

Cystic fibrosis (CF) airway epithelia exhibit raised transepithelial Na+ transport rates, as determined by open-circuit isotope fluxes and estimates of the amiloride-sensitive equivalent short-circuit current (Ieq). To study the contribution of apical and basolateral membrane paths to raised Na+ transport in CF, CF nasal epithelial cultures were studied with double-barreled Na(+)-selective microelectrodes and the Ussing chamber technique. Intracellular Na+ activity (acNa) was 24.1 +/- 1.5 mM (n = 36), a value similar to acNa of normal nasal epithelial cells. Reduction of luminal [Na+] to 3 mM abolished Ieq and reduced acNa. Amiloride (10(-4) M) abolished Ieq but increased acNa from 20 +/- 2 to 36 +/- 7 mM (n = 10). Amiloride-induced increase in acNa was not affected by serosal [Na+] reduction but was blocked by preexposure to reduced luminal [Na+]. Amphotericin B increased Ieq during amiloride exposure, indicating that amiloride did not inhibit NA(+)-K(+)-ATPase. Ouabain abolished Ieq and slowly raised acNa. Reduction of serosal [Na+] led to a decrease in acNa that was blocked by bumetanide. It is concluded that 1) CF airway epithelia exhibit an increased apical membrane Na+ permeability, 2) acNa is regulated to a normal level in CF cells despite increased transcellular Na+ fluxes, 3) the abnormal increase in acNa in response to amiloride is dependent on luminal Na+, 4) Na+ is transported across the basolateral membrane by a bumetanide-sensitive cotransport mechanism, and 5) ouabain inhibits the basolateral Na(+)-K(+)-ATPase, causing slow dissipation of the chemical and electrical gradients across the cell membranes.

After intravenous injection of synthetic 3H-Lys8-vasopressin to intact rats and rats in which the liver and kidneys had been excluded from the circulation, the tissue water to plasma water ratios of radioactivity were determined, usually 12 min after injection, for the following tissues: The adenohypophysis, the neurohypophysis, the median eminence, parts of the hypothalamus containing the supraoptic nuclei, and pieces of the cerebellar cortex. The results were compared with those obtained in similar experiments using 3H-mannitol.Radioactivity could not be shown to accumulate in any part of the hypothalamo-hypophyseal system. Radioactivity disappeared from the plasma in the rats with excluded liver and kidneys at a rate not strikingly different from that in intact rats.In separate experiments, identical in procedure, radiochromatography was performed on plasma samples. The ratio of 3H-tyrosine to 3H-Lys8-vasopressin was about 1 already two min after the injection, both in intact and operated rats. These results seem to indicate a considerable extra-abdominal breakdown of 3H-Lys8-vasopressin, and demonstrate that without special precautions, measurements of tissue and plasma radioactivities cannot be taken as indicators of labelled hormone, even when the organs which have been considered to be the major site of hormone breakdown have been excluded from the circulation.

Inactivation of antidiuretic hormone by slices and homogenate from rat kidney inner medulla was inhibited during a 2 hour exposure to urea in concentrations over 0.5 M. Guanidine and thiourea in equimolar concentrations had an inhibitory effect which was 2 and 1.6 times more marked, respectively, than that of urea. The possible implications of these findings for the function of the concentrating mechanism of the kidneys during different states of hydration are discussed.

Increasing the calcium concentration 5 times or more in the medium used for studying the inactivation of arginine-vasopressin by rat kidney medulla slices caused a marked inhibition of the inactivating activity of such slices. This effect was not found in homogenates of rat kidney medulla. The results are in agreement with the interpretation that the high calcium concentration decreased the cellular permeability to the hormone. This would seem to give a rational explanation of the vasopressin-resistant diabetes insipidus which is found in hypercalcaemia.

A method was developed for studying the inactivation of antidiuretic hormone in slices from different zones of the rat kidney. The essential features of the method are: The use of thin slices from 4 zones: outer cortex, inner cortex, outer medulla and papilla which are incubated aerobically with the hormone in a medium that has a composition essentially the same as rat extracellular fluid, including a calcium concentration of 2.5 mmol.The inactivation of arginine- and lysine-vasopressin by these slices at 25° C and 37° C for 2 hours was studied. All zones inactivated both vasopressins, but the most marked inactivation activity per mg dry tissue was found in the papillary zone. High concentrations of sodium chloride in the incubation medium did not affect the inactivation of vasopressin by papillary slices.The amounts of arginine- and lysine-vasopressin (expressed in rat pressor units) which were inactivated per mg dry tissue in the different zones during the incubation period were equivalent. Similar results were found in experiments on the inactivation of the two hormones by liver slices. These findings are discussed in relation to the problems of the site of action of antidiuretic hormone and the difference in the antidiuretic response to arginine- and lysine-vasopressin which is found after i. v. injection in the rat.