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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.

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.

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.

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.