Cationophore properties of the new polyether antibiotic Salinomycin investigated in distal rabbit colon in vivo and in vitro

The distal rabbit colon was used as a model to investigate the influence of the cationophore Salinomycin in vivo with a single-pass perfusion, and in vitro with a modified Ussing chamber technique. For in vivo experiments with labelled 14C-PEG as a volume marker in the perfusate, a dose of 10E-4 mol/l Salinomycin was used. Net water (53 microliters/h/cm), net chloride (3 mumol/h/cm) and net sodium (3.6 mumol/h/cm) absorption was not significantly influenced, but net potassium secretion (-3 mumol/h/cm) was decreased to zero and transepithelial potential (PD) reduced from -45 mV to -33 mV. 10E-4 mol/l Salinomycin, applied in vitro on the muscosal side, decreased PD in 80 min and 10E-3 mol/l in 30 min from 18 mV to zero. Both concentrations decreased the short-circuit current (Isc = 77 microA/cm2) in 60 min, respectively 30 min to 40 microA/cm2. After 60 min mucosal 10E-4 mol/l Salinomycin the Isc increased, resulting from a transepithelial conductance (Gt) increase from 3 to 40 mS/cm2. A dose-related effect was present on PD, Isc and Gt at concentrations between 10E-7 and 10E-6 mol/l. The unidirectional 22Na fluxes were increased to 20 times the control values and net Na transport disappeared. We conclude that Salinomycin when applied in usual doses (10E-4 mol/l) as a coccidiostatic feed additive, profoundly affects colonic electrolyte transport.     

The cytoskeleton and rat granulosa cell steroidogenesis: possible involvement of microtubules and microfilaments

The participation of both microtubules and microfilaments in granulosa cell steroidogenesis was assessed by monitoring the effects of colchicine (0-250 microM) and/or cytochalasin B (0-10 micrograms/ml) or dihydrocytochalasin B (0-2.0 micrograms/ml) on cellular morphology and production of progestins during 24 h of culture. Both colchicine and the cytochalasins increased granulosa cell production of progesterone and of 20 alpha-hydroxy-pregn-4-en-3-one (20 alpha-OH-progesterone) in a dose-dependent manner. The largest increase in steroidogenesis (about 2- to 3-fold) was observed at 4-250 microM colchicine and at 2-10 micrograms/ml cytochalasin. Those concentrations of the inhibitors of microtubule or microfilament polymerization that stimulated basal progestin production also markedly influenced cell spreading. Whereas cells cultured for 24 h in medium alone became very flattened with numerous cytoplasmic extensions, those cultured with colchicine (0.2-250 microM) or cytochalasin (0.4-2 micrograms/ml) were much less spread and progressively became more rounded and regular in outline. These changes in cell morphology were reflected by decreases in the mean area occupied by the cells on the culture surface of up to 60-65% and reductions in mean contour index values from 5.7 +/- 0.1 (control) to 3.9 +/- 0.1 (250 microM colchicine), 4.2 +/- 0.1 (2 micrograms/ml cytochalasin B), or 4.1 +/- 0.1 (2 micrograms/ml dihydrocytochalasin B). Cultures containing both colchicine and cytochalasin B exhibited a greater steroidogenic response than that elicited by either inhibitor alone. For example, granulosa cell progesterone production was stimulated almost 2-fold by 4 microM colchicine or 2 microM/ml cytochalasin B, but 5.5-fold by 4 microM colchicine plus 2 micrograms/ml cytochalasin B.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uncoupled basal sodium absorption and chloride secretion in prairie dog (Cynomys ludovicianus) gallbladder.

1. Prairie dog gallbladders mounted in a Ussing-type chamber and bathed with symmetrical Ringer's solutions exhibited a transepithelial resistance (Rt) of 51 +/- 5 omega cm2, a lumen negative potential difference (Vms) of 11.5 +/- 0.7 mV and a short-circuit current (Isc) of 6.9 +/- 0.3 microEq/hr/cm2. 2. Radioisotopic ion flux experiments revealed that the basal Isc of 6.9 +/- 0.3 microEq/hr/cm2 was mostly accounted for by net Na+ absorption of 3.2 +/- 0.5 microEq/hr/cm2 and net Cl- secretion of 2.9 +/- 0.3 microEq/hr/cm2. 3. In HCO3- free Ringer's, net Na+ flux was virtually abolished, net Cl- flux decreased by 50% and Isc was reduced by 77%. 4. 10(-3) M mucosal amiloride and DIDS reduced Isc by 28 and 24%, respectively. 5. Mucosal NaCl diffusion potentials indicated that the paracellular pathway was cation selective. 6. Thin section electron micrographs showed a single cell population in this epithelium suggesting that net Na+ absorption and Cl- secretion may emerge from the same cells. 7. We conclude that prairie dog gallbladder epithelium is an electrogenic tissue and, in contrast to gallbladders of most other species, simultaneously but independently absorbs Na+ and secretes Cl-.     

Effects of saline infusion and acute metabolic acidosis and alkalosis on water and electrolyte transport in the human colon.

Both the kidney and colon secrete bicarbonate and transport water and electrolytes. The respective contributions of these two organs to acid-base and electrolyte balance in normal man has thus been studied in eight healthy male volunteers who underwent simultaneous renal clearance studies, and colonic perfusion with a 0.9% saline or 7.2% mannitol solution, during metabolic alkalosis and acidosis, extracellular volume expansion, and control conditions. There was no influence of these acid-base conditions on electrolyte transport in the colon. In the urine, preferential loss of chloride over sodium averaged 81, 143 (P less than 0.001), and 141 (P less than 0.05) muequiv./min, during control, metabolic acidosis, and extracellular volume expansion conditions, respectively. During alkalosis more sodium than chloride was lost (146 muequiv./min) (P less than 0.001). Colonic pH averaged 7.41 during saline and 6.75 (P less than 0.005) during mannitol perfusion. Titratable acid was not produced in the colon during saline perfusion, and averaged 18 muequiv./min during mannitol perfusion. Urinary titratable acid increased from 19 to 25 muequiv./min (P less than 0.01) during volume expansion. With saline perfusion, bicarbonate secretion rate in the colon rose from 249 muequiv./min during control conditions to 289 muequiv./min during metabolic alkalosis (P less than 0.05). More bicarbonate was excreted in the urine during alkalosis when mannitol was introduced in the colon (243 muequiv./min) than when saline was perfused (152 muequiv./min) (P less than 0.05). This study indicates that the response of the human colon is trivial compared with that of the kidney during acute changes in acid-base balance.     

Stimulation of canine pancreatic polypeptide, gastrin, and gastric acid secretion by ranatensin, litorin, bombesin nonapeptide and substance P

Four dogs with chronic gastric fistulas were give intravenous bombesin nonapeptide (B9), ranatensin, and litorin by constant infusion for 90 min at 1.2 micrograms x kg-1 on separate days. A dose response study with substance P (1.5, 3.0, 60, 18 and 54 micrograms x kg-1 x h-1) was also carried out and all tests compared to a standard protein meal (10g x kg-1). Plasma gastrin and PP were measured by radioimmunoassay and gastric acid by autobiuret titration. Substance P failed to stimulate gastric acid secretion or release either pancreatic polypeptide (PP) or gastrin. Basal gastrin levels were 8 +/-2 fmol/ml. The peak increment of gastrin released by bombesin was 95 +/- 16, ranatensin 22 +/- 6, litorin 18 +/- 4, and meal 39 +/- 5 fmol/ml. Bombesin caused significantly greater release of gastrin than a meal, litorin or ranatensin (P less than 0.01). Basal gastric secretion was 23 +/- 4 microequiv./min. B9 produced a peak acid secretion of 356 +/- 124 muequiv./min. There was no significant difference between the bombesin-like peptides (P less than 0.01). Basal plasma PP was 38 +/- 12 fmol/ml. B9 produced a peak PP increment of 600 +/- 50, litorin 137 +/- 36, ranatensin 98 +/- 11, and a meal 305 +/- 58 fmol/ml. B9 released significantly more PP than either litorin of ranatensin (P less than 0.01). The different amino acid sequences of the peptides are probably responsible for their potency. The substitution of a penultimate phenylalanine residue in litorin and ranatensin for leucine in bombesin does not prevent PP or gastrin release by bombesin-like peptides. Since bombesin-like peptides are widely distributed in the gastrointestinal tract of man and stimulate both acid and gut hormone secretion, it is possible that they might play a physiological role in the modulation of gastrointestinal function.     

Potassium reaccumulation by isolated frog epidermis

The involvement of potassium in transepithelial sodium transport was tested by studying net potassium reuptake by potassium-depleted frog skin epidermis. Normal potassium content in half-strength Ringer's (0.244 μequiv/mg dry weight) fell 43% after 16 h in K-free medium at 5°C. Reaccumulation, against an electrochemical potential gradient, to 83% of the initial tissue potassium content occurred following incubation for 4 h at 22°C in K-containing medium. Sodium was required in the solution bathing the inside, but not the outside surface of the skin, for net potassium reaccumulation. Ouabain caused an additional potassium loss from potassium-depleted epidermis, but did not have the same effect on potassium-depleted isolatedcells. Procaine, lithium and caffeine completely inhibited, antidiuretic hormone and cyclic AMP may partially inhibit and amiloride had no effect on potassium reaccumulation. In many cases decreases in sodium and water content were found to occur even in the absence of net potassium reaccumulation. The results suggest (1) potassium is actively transported into the epidermis, (2) this transport is not rigidly coupled to sodium extrusion or water loss, (3) potassium uptake is not rigidly coupled to transepithelial sodium transport, or only a small fraction is involved, (4) potassium diffusion is restricted in the extracellular space.     

Arachidonic acid inhibits Cl secretion in cultures of dog tracheal epithelium.

We studied the effects of arachidonic acid (AA) on Cl secretion across primary cultures of dog tracheal epithelium. Cell sheets showed mean values for baseline short-circuit current (Isc) and transepithelial resistance of 33.8 muA/cm2 and 360 omega.cm2 (n = 44). AA (5 x 10(-5) M) added to both sides increased Isc by 27.8 +/- 5.2 muA/cm2 (mean +/- SE, n = 8), and elevated intracellular cAMP levels. In the presence of 5 x 10(-6) M of both indomethacin (INDO) and nordihydroguaiaretic acid (NDGA) (inhibitors of cyclooxygenase and lipoxygenase, respectively), AA reduced Isc by 4.4 +/- 0.6 muA/cm2 (n = 10) without changing cAMP. Both INDO and NDGA were necessary to abolish the Isc increase in response to AA. The effects of AA on Isc were unaffected by amiloride. In the presence of INDO and NDGA, isoproterenol (ISO) raised cAMP and increased Isc by 27.6 +/- 4.3 (transient) and 12.8 +/- 3.2 muA/cm2 (sustained) (n = 9). With AA present as well as INDO and NDGA, the transient and sustained responses to ISO were significantly reduced to 13.2 +/- 2.4 and 3.9 +/- 0.8 muA/cm2 (n = 10), respectively; the increase in cAMP was unaltered. AA approximately halved baseline efflux of 125I from confluent cell sheets in high K medium and reduced the isoproterenol-induced increase in efflux to 20% of control. These data are consistent with the reported inhibitory effect of AA on apical membrane chloride channels.     

Electrolyte transport across rabbit late proximal colon in vitro

The second part of rabbit proximal colon was investigated in vitro under short circuit conditions. Unidirectional sodium and chloride fluxes were measured during the soft faeces period and during the hard faeces period. Rabbit late proximal colon has a potential difference (psi mS) of 4 mV, a tissue conductance (GT) of 10-11 mS/cm2 and a short circuit current (Isc) of 1.5 mueq/cm2 X hr. Under control conditions sodium (2.65 mueq/cm2 X hr) and chloride (0.67 mueq/cm2 X hr) are absorbed. Ouabain abolished psi ms,Isc and the net sodium flux totally, whereas 0.1 mM amiloride only slightly decreased the net sodium flux. No differences in electrical properties and Na,Cl-fluxes were found between the faeces periods. Removal of sodium abolished psi ms and Isc totally, and a high potassium solution depolarized the preparation (psi ms = 0). A linear current-voltage relation characterizes the tissue as an ohmic resistor between -40 and +50 mV, and reveals a slope conductance of 14 mS/cm2 under KCl conditions. We conclude that the transport functions under in vitro conditions differ markedly from the in vivo situation, and that the diurnal differences of electrolyte transport in vivo occur mainly by the involvement of ionic gradients.     

Stimulation of sodium transport by aldosterone and arginine vasotocin in A6 cells

The effects of aldosterone and arginine vasotocin (AVT) on transepithelial Na+ transport of cultured A6 cells were investigated. All experiments were performed with cells grown on Millicell TM culture-plate inserts for a period of 2-4 weeks in defined, serum-free medium. Omitting fetal bovine serum 2 days after seeding the cells on filters did not influence potential difference (PD) development or the hormonal responses tested. The cell layers were placed in an Ussing chamber for short-circuit current (ISC) and transepithelial conductance (G) measurements. Base-line values were (n = 93): PD, 51.0 +/- 0.2 mV (apical side negative); ISC, 14.55 +/- 0.06 microA/cm2; G, 0.306 +/- 0.001 mS/cm2. ISC and G were higher in cells pretreated with 10(-7) M aldosterone for 24 h in the incubator, when compared to controls (ISC, 28 +/- 2 vs. 16 +/- 2 microA/cm2, G, 0.41 +/- 0.04 vs. 0.26 +/- 0.01 mS/cm2, n = 5) and both remained stable for at least 6 h. In cells not treated with aldosterone, 10(-7) M AVT increased ISC within 1 min after addition, producing a maximum ISC within 15 min which then declined to baseline levels over the next 5 h. Addition of AVT to aldosterone-pretreated cells resulted in a significantly greater peak increase in ISC than in non-pretreated cells (change in ISC compared to controls: 8.1 +/- 0.4 vs. 4.9 +/- 0.4 microA/cm2, n = 5, P less than 0.001), indicating a synergistic effect. A dose-response curve for amiloride obtained in the presence of AVT showed that amiloride completely inhibits ISC. Pretreatment of the A6 cells with aldosterone for 24 h shifted the amiloride dose-response curve to the right, as expressed in a doubling of the apparent Ki value (from 0.17 +/- 0.02 to 0.33 +/- 0.04 microM). In conclusion, A6 cells grown in defined, serum-free medium express a greater than additive synergism between aldosterone and AVT in stimulating transepithelial Na+ transport.     

Paths of ion transport across canine fetal tracheal epithelium

Fluid secretion by the fetal sheep lung is thought to be driven by secretion of Cl- by the pulmonary epithelium. We previously demonstrated Cl- secretion by tracheal epithelium excised from fetal dogs and sheep. In this study we characterized the ion transport pathways across fetal canine tracheal epithelium. The transport of Na+ and Cl- across trachea excised from fetal dogs was evaluated from transepithelial electrical properties and isotope fluxes. Under basal conditions the tissues were characterized by a lumen-negative potential difference (PD) of 11 mV and conductance of 5.2 mS/cm2. The short-circuit current (Isc) was 43 microA/cm2 (1.6 mueq.cm-2.h-1). Basal Na+ flows were symmetrical, but net Na+ absorption (1.1 mueq.cm-2.h-1) could be induced by exposure of the luminal surface to amphotericin B (10(-6) M). Bilateral replacement of Na+ reduced Isc by 85%. Replacement of submucosal Na+ or exposure to submucosal furosemide (10(-4) M) reduced net Cl- secretion by 60-70%. Luminal exposure to indomethacin (10(-6) M) induced a 50% decrease in Isc, whereas isoproterenol (10(-6) M) increased Isc by 120%. The properties of the Cl- secretory pathway across fetal dog trachea are consistent with the model proposed for Cl- secretion across adult dog trachea and other Cl- -secreting tissues (e.g., bullfrog cornea and shark rectal gland). The absence of basal Na+ absorption by fetal dog trachea probably reflects limited apical membrane Na+ permeability.     

Prolactin directly stimulated the solvent drag-induced calcium transport in the duodenum of female rats

Prolactin has been reported to stimulate the calcium absorption of the duodenum where three components of the active calcium transport, namely transcellular active, voltage-dependent and solvent drag-induced calcium transport, were identified. It was known that the transcellular active, but not the voltage-dependent, duodenal calcium transport was directly stimulated by prolactin. The present study thus aimed to evaluate the direct action of prolactin on the solvent drag-induced duodenal calcium transport by using the Ussing chamber technique. The jejunum was used as a reference for the existence of solvent drag and the widening of tight junction induced by cytochalasin E. Results showed that the solvent drag-induced calcium transport existed in both intestinal segments, but the magnitude was significantly greater in the duodenum (29.27+/-2.27 vs. 17.31+/-1.65 nmol h(-1) cm(-2), P<0.001). We further demonstrated that 200, 600 and 800, but not 1000 ng/ml, prolactin significantly promoted the solvent drag-induced duodenal calcium transport in a dose-response manner, i.e. from the control value of (nmol h(-1) cm(-2)) 24.31+/-2.36 to 45.42+/-3.47 (P<0.01), 63.82+/-5.28 (P<0.001) and 53.93+/-5.41 (P<0.01), respectively. However, prolactin did not manifest any effect on the jejunum. Because the paracellular transport was suggested to be size-selective as well as charge-selective, further experiments were designed to evaluate the mechanism by which prolactin stimulated the solvent drag-induced calcium transport. The duodenum was exposed to 20 microM cytochalasin E, 600 ng/ml prolactin or the combination of both in the presence of a paracellular marker 3H-mannitol, while the jejunum was a positive reference. The results showed that, in the jejunum, cytochalasin E alone and cytochalasin E plus prolactin significantly increased the mannitol fluxes from (micromol h(-1) cm(-2)) 0.29+/-0.04 to 0.49+/-0.03 (P<0.05) and 0.48+/-0.05 (P<0.05), respectively, while having no effect on the calcium fluxes. Prolactin alone had no effect on the jejunal calcium flux. In the duodenum, neither mannitol nor calcium fluxes were enhanced by cytochalasin E, however, prolactin still increased the solvent drag-induced calcium flux from 27.74+/-2.41 to 51.03+/-4.35 nmol h(-1) cm(-2) (P<0.001). It was concluded that prolactin directly stimulated the solvent drag-induced duodenal calcium transport in a dose-response and biphasic manner without the widening of tight junction.     

Ion transport in an immortalized rat submandibular cell line SMG-C6

The immortalized rat submandibular epithelial cell line, SMG-C6, cultured on porous tissue culture supports, forms polarized, tight-junction epithelia facilitating bioelectric characterization in Ussing chambers. The SMG-C6 epithelia generated transepithelial resistances of 956+/-84Omega.cm2 and potential differences (PD) of -16.9 +/- 1.5mV (apical surface negative) with a basal short-circuit current (Isc) of 23.9 +/- 1.7 microA/cm2 (n = 69). P2 nucleotide receptor agonists, ATP or UTP, applied apically or basolaterally induced a transient increase in Isc, followed by a sustained decreased below baseline value. The peak DeltaIsc increase was partly sensitive to Cl- and K+ channel inhibitors, DPC, glibenclamide, and tetraethylammonium (TEA) and was completely abolished following Ca2+ chelation with BAPTA or bilateral substitution of gluconate for Cl-. The major component of basal Isc was sensitive to apical Na+ replacement or amiloride (half-maximal inhibitory concentration 392 nM). Following pretreatment with amiloride, ATP induced a significantly greater Isc; however, the poststimulatory decline was abolished, suggesting an ATP-induced inhibition of amiloride-sensitive Na+ transport. Consistent with the ion transport properties found in Ussing chambers, SMG-C6 cells express the rat epithelial Na+ channel alpha-subunit (alpha-rENaC). Thus, cultured SMG-C6 cells produce tight polarized epithelia on permeable support with stimulated Cl- secretory conductance and an inward Isc accounted for by amiloride-sensitive Na+ absorption.     

Stimulation by aldosterone of a conductive chloride pathway in toad skin

As a rule, chloride movement (JC1-) across amphibian skin is considered to be passive; this is implied in fact for preparations incubated in Ringer's fluid, since short-circuit current (Isc) is the quantitative expression of net, active sodium transport (JNa+). The nature of the Cl- pathway(s) was investigated by incubating amphibian skin (mostly Bufo marinus) with Cl- present on the epithelial side only, and after blocking JNa+ by combined treatment with ouabain and amiloride. In such conditions, JCl- was found to be equal to (reversed) Isc; furthermore, when JCl- was "translated" in terms of conductance, gCl-, the latter accounted almost quantitatively for transepithelial conductance, g1. When residual intratissue (i.e. intracellular) electronegativity was eliminated by replacing Na+ with K+, JCl- was larger but Isc and JCl- were still found to reflect each other, and gCl- again accounted for most, if not all, of g1. JCl- in the opposite direction, as a result of Cl- being present only on the dermal side, was negligible, and g1 was very low. Thus, in the absence of sodium transport, when experimental conditions are such that a net inward JCl- obtains, the anion apparently flows only through (a) conductive pathway(s). Aldosterone is probably involved in the regulation of this pathway, as JCl- was much lower when toads were maintained in dilute saline than in water or on moist peat; so was the fraction of the apical surface corresponding to mitochondria-rich cells.     

In vitro differentiated HT 29-D4 clonal cell line generates leakproof and electrically active monolayers when cultured in porous-bottom culture dishes

HT 29-D4 is a clonal cell line, derived from the human colon adenocarcinoma cell line HT 29, which can be induced to differentiate into enterocyte-like cells by replacing glucose with galactose in the culture medium (Fantini et al. [1986], J. Cell Sci. 83, 235-249). Both undifferentiated and differentiated HT 29-D4 cells have been successfully grown to confluency in Costar Transwell permeable chambers. Only HT 29-D4 cells grown in glucose-free, galactose-containing medium were able to form leakproof monolayers, as demonstrated by their ability to prevent diffusion of serum proteins. These monolayers consist of highly polarized epithelial-like cells with a well organized apical brush border. Transepithelial electrical parameters have been measured under sterile conditions for both types of monolayer. Only HT 29-D4 monolayers cultured in glucose-free, galactose-containing medium were electrically active, with a transepithelial resistance R = 172 +/- 46 omega.cm2, a potential difference PD = 0.35 +/- 0.05 mV, apical negative and a short-circuit current Isc = 2.0 +/- 0.4 microA.cm-2. Apical addition of amphotericin B induced a rapid and considerable increase in Isc and PD, which was abolished by basal ouabain. In contrast, HT 29-D4 cells grown in glucose-containing medium did not generate any potential difference (PD = 0 mV) and their resistance was very low (R = 34.1 +/- 0.9 omega.cm2). It is concluded from these studies that HT 29-D4 cells grown in glucose-free, galactose-containing medium acquire functional characteristics of epithelia, compared to HT 29-D4 cells grown in glucose-containing medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Avian colonic ion transport: effects of corticosterone and dexamethasone

1. Corticosterone, a natural corticosteroid hormone in birds, when injected into domestic fowl (Gallus domesticus) (2000 micrograms.kg-1, 4-5 h before experiment) increases both the basal Isc (short-circuit current) and amiloride-sensitive Isc as well as the PD across the colon in vitro. Dexamethasone, a synthetic analogue (650 micrograms.kg-1, 4-5 h before experiment) also increases the basal and amiloride-sensitive Isc as well as PD in these preparations. 2. In marked contrast, longer term injection or infusion of dexamethasone (650 micrograms.kg-1) for 3 or more days caused a decline in basal Isc and PD (the PD often reversed with the serosal side becoming electronegative) and a drop in resistance. However in these preparations, the amiloride-sensitive Isc was significantly elevated which could be accounted for by an increase in net Na flux. 3. No significant change occurs in net flux of Cl or K although unidirectional fluxes in both directions were increased for both ions in birds given dexamethasone for 3 days. 4. A disparity between the basal Isc and the amiloride-sensitive Isc appeared in these preparations from dexamethasone injected birds reflecting the transport of other ions, possibly HCO3- or H+. The possible role of corticosterone in mineral metabolism of birds is discussed.     

Impairment of conjunctival glutathione secretion and ion transport by oxidative stress in an adenovirus type 5 ocular infection model of pigmented rabbits.

Conjunctival epithelial cells of pigmented rabbits secrete reduced glutathione (GSH) into the apical (mucosal) fluid. The aim of the current study was to determine the effect of oxidative stress resulting from viral infection and that of GSH supplementation on redox status, GSH, and ion transport in freshly excised conjunctival tissues and epithelial cell layers in primary culture (RCEC) of adenovirus type 5 (Ad5)-infected rabbits. Lipid peroxidation (LPO) products, nitric oxide (NO), and expression of nitric oxide synthase (NOS2) were quantitated as a function of time after viral inoculation. Unidirectional fluxes of [3H]GSH and changes in short-circuit current (Isc) from mucosal supplementation of Ad5-inoculated conjunctival tissues with GSH and glutathione monoethyl ester (GSH-MEE) were also measured. Ad5 inoculation significantly decreased conjunctival GSH level by 19, 45, 48, and 50% at 8, 24, 48, and 72 h postinfection, respectively. LPO product and NO levels increased significantly (2- and 100-fold, respectively) above that of uninfected controls on Day 3 post-Ad5 inoculation, and co-treatment with GSH-MEE and tocopherol succinate abolished this effect. NO levels showed a progressive increase post-Ad5 inoculation, reaching 0.22 +/- 0.06, 8.12 +/- 0.91, and 2.05 +/- 0.65 microM on Days 1, 3, and 5, respectively, and the highest level was observed on the day of maximal viral replication (Day 3). A very significant induction of the expression of NOS2 on Days 1, 3, and 5 post-Ad5 inoculation was observed. Uninfected control conjunctival tissues displayed a net serosal-to-mucosal GSH flux (Jsm), where the mucosal-to-serosal flux (Jms) was approximately 14 pmol h(-1) cm(-2) and the Jsm was approximately 22 pmol h(-1) cm(-2). In Ad5-inoculated rabbits similar GSH flux was observed in both the sm and ms directions, and the net GSH flux was negligible. Isc and potential difference (PD) across conjunctival tissues of Ad5-inoculated rabbits decreased by > or = 50% compared with control, while the transepithelial electrical resistance (TEER) remained unchanged. Mucosal, but not serosal, superfusion of GSH or GSH-MEE in Ad5-inoculated conjunctival tissues increased the Isc by up to 40% in approximately 100 min. Our results show that net secretion of GSH across rabbit conjunctiva is totally blocked after Ad5 inoculation and active ion transport rate decreased by approximately 50%. Decreased net GSH secretion into mucosal fluid after Ad5 infection may have resulted from a decreased intracellular GSH pool due to oxyradical-induced changes in redox status and lower active ion transport. Mucosal treatment of Ad5-infected conjunctival tissues with pharmacological levels of GSH appears to transstimulate mucosal GSH secretion and restore active ion transport activity, suggesting a potentially useful therapeutic regimen for ocular infections.     

Evaluation of SQ 28,603, an inhibitor of neutral endopeptidase, in conscious monkeys.

The potent neutral endopeptidase inhibitor SQ 28,603 (N-(2-(mercaptomethyl)-1-oxo-3-phenylpropyl)-beta-alanine) significantly increased excretion of sodium from 4.9 +/- 2.3 to 14.3 +/- 2.1 muequiv./min and cyclic 3',5'-guanosine monophosphate from 118 +/- 13 to 179 +/- 18 pmol/min after intravenous administration of 300 mumol/kg (approximately 80 mg/kg) in conscious female cynomolgus monkeys. SQ 28,603 did not change blood pressure or plasma atrial natriuretic peptide concentrations in the normal monkeys. In contrast, 1-h infusions of 3, 10, or 30 pmol.kg-1.min-1 of human atrial natriuretic peptide lowered blood pressure by -3 +/- 4, -9 +/- 4, and -27 +/- 3 mmHg (1 mmHg = 133.322 Pa), increased cyclic guanosine monophosphate excretion from 78 +/- 11 to 90 +/- 6, 216 +/- 33, and 531 +/- 41 pmol/min, and raised plasma atrial natriuretic peptide from 7.2 +/- 0.7 to 21 +/- 4, 62 +/- 12, and 192 +/- 35 fmol/mL without affecting sodium excretion. In monkeys receiving 10 pmol.kg-1.min-1 of atrial natriuretic peptide, 300 mumol/kg of SQ 28,603 reduced mean arterial pressure by -13 +/- 5 mmHg and increased sodium excretion from 6.6 +/- 3.2 to 31.3 +/- 6.0 muequiv./min, cyclic guanosine monophosphate excretion from 342 +/- 68 to 1144 +/- 418 pmol/min, and plasma atrial natriuretic peptide from 124 +/- 8 to 262 +/- 52 fmol/mL. In conclusion, SQ 28,603 stimulated renal excretory function in conscious monkeys, presumably by preventing the degradation of atrial natriuretic peptide by neutral endopeptidase.     

Passive and active transport properties of a gill model, the cultured branchial epithelium of the freshwater rainbow trout (Oncorhynchus mykiss)

Branchial epithelia of freshwater rainbow trout were cultured on permeable supports, polyethylene terephthalate membranes ("filter inserts"), starting from dispersed gill epithelial cells in primary culture. Leibowitz L-15 media plus foetal bovine serum and glutamine, with an ionic composition similar to trout extracellular fluid, was used. After 6 days of growth on the filter insert with L-15 present on both apical and basolateral surfaces, the cultured preparations exhibited stable transepithelial resistances (generally 1000-5000 ohms cm2) typical of an electrically tight epithelium. Under these symmetrical conditions, transepithelial potential was zero, and unidirectional fluxes of Na+ and Cl- across the epithelium and permeability to the paracellular marker polyethylene glycol-4000 (PEG) were equal in both directions. Na+ and Cl- fluxes were similar to one another and linearly related to conductance (inversely related to resistance) in a manner indicative of fully conductive passive transport. Upon exposure to apical fresh water, transepithelial resistance increased greatly and a basolateral-negative transepithelial potential developed. At the same time, however, PEG permeability and unidirectional effluxes of Na+ and Cl- increased. Thus, total conductance fell, and ionic fluxes and paracellular permeability per unit conductance all increased greatly, consistent with a scenario whereby transcellular conductance decreases but paracellular permeability increases upon dilution of the apical medium. In apical fresh water, there was a net loss of ions from the basolateral to apical surfaces as effluxes greatly exceeded influxes. However, application of the Ussing flux ratio criterion, in two separate series involving different methods for measuring unidirectional fluxes, revealed active influx of Cl- against the electrochemical gradient but passive movement of Na+. The finding is surprising because the cultured epithelium appears to consist entirely of pavement-type cells.     

Optical measurement of osmotic water transport in cultured cells. Role of glucose transporters

Methodology was developed to measure osmotic water permeability in monolayer cultured cells and applied to examine the proposed role of glucose transporters in the water pathway (1989. Proc. Natl. Acad. Sci. USA. 86:8397-8401). J774 macrophages were grown on glass coverslips and mounted in a channel-type perfusion chamber for rapid fluid exchange without cell detachment. Relative cell volume was measured by 45 degrees light scattering using an inverted microscope; measurement accuracy was validated by confocal imaging microscopy. The time required for greater than 90% fluid exchange was less than 1 s. In response to a decrease in perfusate osmolality from 300 to 210 mosM, cells swelled without lag at an initial rate of 4.5%/s, corresponding to a water permeability coefficient of (6.3 +/- 0.4) x 10(-3) cm/s (SE, n = 20, 23 degrees C), assuming a cell surface-to-volume ratio of 4,400 cm-1. The initial rate of cell swelling was proportional to osmotic gradient size, independent of perfusate viscosity, and increased by amphotericin B (25 micrograms/ml), and had an activation energy of 10.0 +/- 1 kcal/mol (12-39 degrees C). The compounds phloretin (20 microM) and cytochalasin B (2.5 micrograms/ml) inhibited glucose transport by greater than 85% but did not influence Pf in paired experiments in which Pf was measured before and after inhibitor addition. The mercurials HgCl2 (0.1 mM) and p-chloromercuribenzoate (1 mM) did not inhibit Pf. A stopped-flow light scattering technique was used to measure Pf independently in J774 macrophages grown in suspension culture. Pf in suspended cells was (4.4 +/- 0.3) x 10(-3) cm/s (assuming a surface-to-volume ratio of 8,800 cm-1), increased more than threefold by amphotericin B, and not inhibited by phloretin and cytochalasin B under conditions of strong inhibition of glucose transport. The glucose reflection coefficient was 0.98 +/- 0.03 as measured by induced osmosis, assuming a unity reflection coefficient for sucrose. These results establish a quantitative method for measurement of osmotic water transport in adherent cultured cells and provide evidence that glucose transporters are not involved in the water transporting pathway.     

Heptaminol hydrochloride as an epithelium transporter

Electrophysiological and transport effects induced by heptaminol hydrochloride were studied in frog epithelium. This tissue, which can easily be maintained in vitro, is a valuable model for studying sodium active transport with hormone-dependent characteristics that reproduce mammalian nephron behavior (notably in areas with tight gap junctions). The two following techniques were used: the Ussing short-circuit current method and the swept-frequency impedance measurement method. Our findings indicate the following. (i) Heptaminol hydrochloride significantly increases the short-circuit current and transepithelial polarization. (ii) This effect develops progressively as the molecule is introduced on the serous side (3Na+/2K+ active countertransport sites). Time to maximum development is approximately 20 min and the electrophysiological effect lasts from 60 to 90 min. (iii) The mean equivalent cationic current rise is larger in sulfate-Ringer (+23 +/- 4.6 microA, p less than 0.01) than in chloride-Ringer (+14 +/- 4.9 microA, p less than 0.05). The increase in short-circuit current is approximately 0.9 muequiv. cm-2 h-1 in sulfate-Ringer. (iv) The increase in mean polarization is greater in chloride (+21 +/- 6.2 mV, p less than 0.02) than in sulfate (+6 +/- 1.5 mV, p less than 0.01) following a diphasic effect on potential. (v) Changes in apical impedance Z are small (-454 +/- 323 omega, nonsignificant) compared with transepithelial resistance in sulfate (-1065 +/- 359 omega, p less than 0.05). (vi) Changes in membrane capacitance reflect changes in the membrane surface. However, no significant capacitance changes are produced in sulfate and chloride solution by heptaminol hydrochloride (-0.04 +/- 0.11 microF and 0.05 +/- 0.11 microF, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)