Depressive symptom reversal for women in a primary care setting: a pilot study

Our goal is to assess the viability of an in vitro preparation of bovine ciliary body/epithelium (CBE) in a small volume Ussing-type chamber. A new small volume Ussing-type chamber with continuous perfusion was developed for bovine CBE. The trans-CBE electrical parameters were monitored and the electrical responses of the CBE to ouabain (1 and 0.01 mM) were recorded. The trans-CBE fluxes of [14C]-L-ascorbate and [3H]-L-glucose were also studied. The bovine CBE preparation was stable inside the chamber in terms of its potential difference (PD), short circuit current (SCC) and trans-CBE resistance. They were -0.51+/-0.05 mV (aqueous side negative), -5.43+/-0.04 microAcm-2 and 94+/-2 Q.cm2 (mean s.e.m., n=35), respectively. The preparation hyperpolarised when 0.01 mM ouabain was administered to the aqueous side, depolarised when ouabain was applied to the stromal side. [3H]-L-glucose diffusion was about 74 nEq h(-1)cm(-2) in either direction (n=12). Taking the area magnification factor of the CBE into consideration, the diffusional L-glucose flux across the bovine CBE was comparable to other tight epithelia. A significant net ascorbate flux (0.26+/-0.05 nEq h(-1)cm(-2), n=4, p<0.01) was found in the stroma to aqueous direction. We have developed a viable in vitro bovine CBE preparation which was (1) electrically stable, (2) responsive to ouabain, (3) tight to L-glucose diffusion, and (4) capable of actively secreting ascorbate. A net trans-CBE chloride transport (0.81+/-0.30 microEq h(-1)cm(-2), n=12, p=0.01) from stromal to aqueous side was found in the present in vitro model under short-circuited conditions.     

Effects of HCO3- on cell composition of rabbit ciliary epithelium: a new model for aqueous humor secretion

PURPOSE: To determine whether the Na+-K+-2Cl- symport or the parallel Na+/H+ and Cl-/HCO3- antiports provide the dominant pathway for NaCl uptake into the ciliary epithelium. Both pathways are known to support NaCl entry from the stroma into the pigmented ciliary epithelial (PE) cells, after which Na+ and Cl- diffuse across the gap junctions into the nonpigmented ciliary epithelial (NPE) cells and are released into the aqueous humor. METHODS: Rabbit iris ciliary bodies were preincubated in HCO3-/CO2-containing or HCO3-/CO2-free solutions before quick freezing, cryosectioning, dehydration, and electron probe x-ray microanalysis. RESULTS: The NPE and the PE cells contained more K and Cl when incubated with bicarbonate. Inhibition of carbonic anhydrase with 0.5 mM acetazolamide had little effect in HCO3--free medium but prevented the increase in Cl in both cell types in HCO3-/CO2 solution. Inhibition of the Na+-K+-2Cl- symport with 10 to 500 microM bumetanide caused Cl loss from both cell types in HCO3--free solution, but bumetanide produced a paradoxical increase in Cl and Na in HCO3-/CO2 solution. Together, acetazolamide and bumetanide resulted in significant Cl loss in HCO3--free solution and prevented the gains of Cl and Na in HCO3-/CO2 solution. CONCLUSIONS: The present results indicate that the dominant entry pathway of NaCl from the stroma into the ciliary epithelial syncytium is through an acetazolamide-inhibitable Cl-/HCO3 and a parallel Na+/H+ antiport. The dominant release pathways into the aqueous humor appear to be a Na+-K+-2Cl-symport, which can be outwardly directed under physiological conditions, together with the Na+/K+-exchange pumps and Cl- channels.     

Complement activation of electrogenic ion transport in isolated rat colon

The complement cascade is an important component in many immune and inflammatory reactions and may contribute to both the diarrhoea and inflammation associated with inflammatory bowel disease. Isolated rat colonic mucosae were voltage clamped in Ussing chambers. Basolateral addition of zymosan-activated whole human serum (ZAS) induced a rapid onset, transient inward short circuit current (SCC). This response was concentration dependent and was significantly attenuated by pre-heating ZAS at 60 degrees C for 30 min. Depletion of complement from normal human serum with cobra venom factor (CVF) significantly lowered SCC responses. Chloride was the primary charge carrying ion as responses to ZAS were abolished in the presence of the loop diuretic bumetanide. The complement component C3a stimulated ion transport but not to the same extent as whole serum. Exogenous C5 was without effect. The cyclooxygenase inhibitor piroxicam significantly attenuated the response to ZAS. These findings support the possibility that complement activation may contribute to the pathophysiology of secretory diarrhoea since activation of electrogenic chloride secretion converts intestinal epithelia to a state of net fluid secretion.     

Role for anions in pulmonary endothelial permeability

beta-Adrenergic stimulation reduces albumin permeation across pulmonary artery endothelial monolayers and induces changes in cell morphology that are mediated by Cl- flux. We tested the hypothesis that anion-mediated changes in endothelial cells result in changes in endothelial permeability. We measured permeation of radiolabeled albumin across bovine pulmonary arterial endothelial monolayers when the extracellular anion was Cl-, Br-, I-, F-, acetate (Ac-), gluconate (G-), and propionate (Pr-). Permeability to albumin (Palbumin) was calculated before and after addition of 0.2 mM of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), which reduces permeability. In Cl-, the Palbumin was 3.05 +/- 0.86 x 10(-6) cm/s and fell by 70% with the addition of IBMX. The initial Palbumin was lowest for Pr- and Ac-. Initial Palbumin was higher in Br-, I-, G-, and F- than in Cl-. A permeability ratio was calculated to examine the IBMX effect. The greatest IBMX effect was seen when Cl- was the extracellular anion, and the order among halide anions was Cl- > Br- > I- > F-. Although the level of extracellular Ca2+ concentration ([Ca2+]o) varied over a wide range in the anion solutions, [Ca2+]o did not systematically affect endothelial permeability in this system. When Cl- was the extracellular anion, varying [Ca2+]o from 0.2 to 2.8 mM caused a change in initial Palbumin but no change in the IBMX effect. The anion channel blockers 4-acetamido-4'-isothiocyanotostilbene-2, 2'-disulfonic acid (0.25 mM) and anthracene-9-carboxylic acid (0.5 mM) significantly altered initial Palbumin and the IBMX effect. The anion transport blockers bumetanide (0.2 mM) and furosemide (1 mM) had no such effects. We conclude that extracellular anions influence bovine pulmonary arterial endothelial permeability and that the pharmacological profile fits better with the activity of anion channels than with other anion transport processes.     

Stimulation of intracellular chloride accumulation by noradrenaline and hence potentiation of its depolarization of rat arterial smooth muscle in vitro

1. Double-barrelled ion-selective microelectrodes were used to examine the effects of exogenous noradrenaline upon the membrane potential (Em) and intracellular chloride concentration ([Cl]i) of arterial smooth muscle from the saphenous branch of the femoral artery of the rat. 2. After treatment with 0.6 mM 6-hydroxydopamine (to functionally denervate the tissue), exogenous noradrenaline (5 nM) caused repeatable depolarization of Em from -63.7 +/- 2.4 mV (s.d., n = 18) to -53.8 +/- 3.4 mV (P < 0.0001) and increases in [Cl]i from 31.0 +/- 0.5 mM to 42.5 +/- 2.2 mM (P < 0.0001). 3. In the presence of 10 microM bumetanide (an inhibitor of (Na-K-Cl) cotransport), 5 nM noradrenaline caused a depolarization of Em of 3.0 +/- 3.2 mV, and a rise in [Cl]i of 4.5 +/- 2.5 mM. 4. In the presence of bumetanide and 1 mM acetazolamide (used as an inhibitor of a Na-independent inward Cl pump), noradrenaline had no effect on Em or [Cl]i. 5. In the absence of extracellular chloride, the rise in apparent [Cl]i in response to 5 nM noradrenaline was abolished but there was a depolarization of 2.0 +/- 3.9 mV. 6. These results are consistent with the stimulation of (Na-K-Cl) cotransport and a Na-independent Cl pump by exogenous noradrenaline and with the consequent increase in [Cl]i and shift in ECl potentiating the depolarization caused by noradrenaline. The possibility that modulation of [Cl]i may be a general mechanism of Em regulation is discussed.     

Mechanism of sodium and chloride transport in the thin ascending limb of Henle

Our previous in vitro studies have disclosed that the thin ascending limb of Henle (tALH) possesses some unique membrane characteristics. In those studies we failed to demonstrated active transport of sodium chloride by the tALH, although it was shown that the isotopic permeability to sodium and chloride was unusually high. However, we did not examine the mechanisms by which the apparent high permeation of sodium chloride occurs. Thus the purpose of the present studies was to elucidate the mechanism of sodium chloride transport across the isolated tALH of the rabbit by conducting four different types of studies: (1) comparison of the observed chloride and sodium flux ratios to those predicted by Ussing's equation under imposed salt concentration gradients; (2) kinetic evaluation of chloride and sodium fluxes; (3) examination of the effect of bromide on the kinetics of chloride transport; and (4) experiments to test for the existence of exchange diffusion of chloride. In the first set of studies the predicted and the theoretical flux ratios of sodium were identical in those experiments in which sodium chloride was added either to the perfusate or to the bath. However, the observed chloride flux ratio, lumen-to-bath/bath-to-lumen, was significantly lower than that predicted from Ussing's equation when 100 mM sodium chloride was added to the bath. In the second set of experiments the apparent isotopic permeability for sodium and for chloride was measured under varying perfusate and bath NaCl concentrations. There was no statistical change in the apparent sodium permeability coefficient when the NaCl concentration was raised by varying increments from 85.5 to 309.5 mM. However, permeation of 36Cl decrease significantly with an increase in Cl from 73.6 to 598.6 mM. These events could be explained by a two component chloride transport process consisting of simple diffusion and a saturable facilitated diffusion process with a Vmax = 3.71 neq mm-1 min-1. In the third set of studies it was shown that bromide inhibits transport of chloride and that the magnitude of inhibition is dependent on chloride concentrations. The fourth set of studies ruled out the existence of exchange diffusion. In conclusion, these studies indicate that sodium transport across tALH is by simple passive diffusion, while chloride transport across tALH involves at least two mechanisms: (1) simple passive diffusion; and (2) a specific membrane interaction process (carrier-mediated) which is competitively inhibited by bromide.     

Sodium chloride transport across the chicken coprodeum. Basic characteristics and dependence on sodium chloride intake

1. The transport characteristics of the chicken coprodeum have been examined in vitro using the isolated mucosa. The short-circuit current (I(sc)), the transepithelial electrical potential difference (p.d.), the unidirectional transmural fluxes (J(ms), J(sm)) of sodium and chloride measured in the short-circuited state, and the unidirectional influx of sodium and chloride across the brush border membrane measured under open-circuit conditions have been studied. The effect of the sodium chloride contents of the diet on these parameters have been investigated.2. The isolated mucosa depends functionally on the presence of glucose in the incubation media. This dependence reflects the need of glucose as a fuel. There is no indication of coupling between transport of sugars and sodium across the brush border membrane. For preparations from chickens on a low sodium diet a very high and stable I(sc) can quantitatively be accounted for by the net transport of sodium. Influx of sodium across the brush border membrane is not significantly different from the net flux of sodium. By feeding the chickens a high sodium diet the I(sc) is reduced by more than 95%, the net transport of sodium is abolished, and the transepithelial electrical conductance is reduced by more than 50%.3. Both unidirectional transepithelial fluxes of chloride, and the serosa to mucosa flux of sodium appear to proceed through a paracellular shunt.4. Under the conditions of the low sodium diet the paracellular pathway appears to be anion selective. Whereas, under the conditions of the high sodium regimen the paracellular route appears to be cation selective. After adaptation to a high sodium diet the influx of sodium across the brush border membrane is only moderately reduced. Consequently the decisive event in the adaptation must be localized elsewhere.     

Transmural ionic fluxes and electrical potential difference in the human jejunum during perfusion with a dihydroxy bile acid

Perfusion studies of the proximal jejunum were performed in healthy volunteers to define the influence of glycochenodeoxycholic acid (GCDC) 2.5 mmol/1 on the net movements of water and electrolytes, the bidirectional fluxes of sodium, potassium, and chloride, and the transmural electrical potential difference (PD). The flux data supported the notion that active sodium transport is inhibited by luminal GCDC, which on the other hand elicits active secretion of chloride. PD was 3 +/- 1 mV, lumen negative, and was not influenced by GCDC. The flux data fit a previously proposed model for the GCDC effect.     

A comparative diuretic and tissue distribution study of bumetanide and furosemide in the dog

Intravenous dose-response data obtained from renal clearance studies in anesthetized dogs indicated that bumetanide was approximately 30-fold more potent than furosemide in enhancing sodium excretion. After the administration of 0.01 mg/kg of bumetanide or 1.0 mg/kg of furosemide, the relationship between i.v. diuretic activity and tissue distribution was evaluated. In dog renal clearance experiments, bumetanide and furosemide significantly enhanced urine flow, sodium and potassium excretion. Inulin clearance as an estimate of glomerular filtration rate was not altered by either drug, but sodium reabsorption was decreased with bumetanide (13%) and furosemide (12%). At these diuretic doses, both compounds were bound to dog plasma protein to about the same extent (86-91%), although total plasma levels were 100-fold higher for furosemide. Within 1/2 hour after the i.v. administration of 14C-bumetanide or 14C-furosemide, 86 to 99% of the 14C in urine, plasma, kidney, and liver appeared as unchanged drug. One minute after maximal diuresis bumetanide was found to have a higher affinity (3-fold) for kidney compared to furosemide. These data offer a possible explanation for the i.v. diuretic potency difference between these two compounds. Furthermore, the lack of significant difference in plasma protein binding and the absence of urinary metabolites of either drug suggest that other factors may also contribute to the marked differences in diuretic activity between bumetanide and furosemide.     

Changes in rat retinal ganglion neuronotrophic factor and its mRNA during postnatal development

It is believed that chloride transport through the macula densa (MD) cells is a factor involved in the tubuloglomerular feedback (TGF) mechanism and in MD-mediated renin release. In this study isolated and perfused rabbit kidney cortical thick ascending limb (cTAL) segments containing MD plaques and attached glomeruli were loaded with chloride (CL-sensitive) 6 methoxy-1-fluorophore (sulphanate-propyl) quinolinium (SPQ). MD and cTAL intracellular chloride concentration ([Cl-]i) was determined by using image-intensified video microscopy and digital image-processing for measuring the intensity of the emitted SPQ fluorescence. With 150 mM NaCl in lumen and bath the [Cl-]i in MD and cTAL cells was 58.8 +/- 7.2 mM (n = 20) and 68.7 +/- 9.8 mM (n = 14), respectively. When the presumed luminal Na(+)-2Cl(-)-K+ co-transporter was blocked by adding 10(-4)M furosemide, the [Cl-]i was reduced in both, MD and cTAL cells from 55.5 +/- 11.9 to 28.6 +/- 10.0 mM (n = 10) and from 43.8 +/- 2.6 to 13.1 +/- 4.5 mM (n = 5), respectively. A reduction in luminal NaCl from 150 to 30 mM also decreased both, MD and cTAL [Cl-]i from 69.4 +/- 9.1 to 36.5 +/- 5.1 mM (n = 9) and from 82.9 +/- 14.5 to 49.4 +/- 8.0 mM (n = 8), respectively. Basolateral addition of the Cl(-)-channel blocker NPPB increased MD [Cl-]i from 31.1 +/- 2.0 to 100.7 +/- 17.0 mM (n = 5) and cTAL [Cl-]i from 44.4 +/- 12.9 to 89.7 +/- 11.7 mM (n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolated perfused rabbit colon crypts: stimulation of Cl- secretion by forskolin

The aim of this study was to characterize ion conductances and carrier mechanisms of isolated in vitro perfused rabbit colonic crypts. Crypts were isolated from rabbit colon mucosa and mounted on a pipette system which allowed controlled perfusion of the lumen. In non-stimulated conditions basolateral membrane voltage (Vbl) was -65 +/- 1 mV (n = 240). Bath Ba2+ (1 mmol/l) and verapamil (0.1 mmol/l) depolarized Vbl by 21 +/- 2 mV (n = 7) and 31 +/- 1 (n = 4), respectively. Lowering of bath Cl- concentration hyperpolarized Vbl from -69 +/- 3 to -75 +/- 3 mV (n = 9). Lowering of luminal Cl- concentration did not change Vbl. Basolateral application of loop diuretics (furosemide, piretanide, bumetanide) had no influence on Vbl in non-stimulated crypts. Forskolin (10(-6) mol/l) in the bath depolarized Vbl by 29 +/- 2 mV (n = 54) and decreased luminal membrane resistance. In one-third of the experiments a spontaneous partial repolarization of Vbl was seen in the presence of forskolin. During forskolin-induced depolarization basolateral application of loop diuretics hyperpolarized Vbl significantly and concentration dependently with a potency sequence of bumetanide > piretanide > or = furosemide. Lowering bath Cl- concentration hyperpolarized Vbl. Lowering of luminal Cl- concentration from 120 to 32 mmol/l during forskolin-induced depolarization led to a further depolarization of Vbl by 7 +/- 2 mV (n = 10). We conclude that Vbl of rabbit colonic crypt cells is dominated by a K+ conductance. Stimulation of the cells by forskolin opens a luminal Cl- conductance. Basolateral uptake of Cl- occurs via a basolateral Na+:2Cl-:K+ cotransport system.     

Ouabain-insensitive salt and water movements in duck red cells. III. The role of chloride in the volume response

Duck red cells in hypertonic media experience rapid osmotic shrinkage followed by gradual reswelling back toward their original volume. This uptake of salt and water is self limiting and demands a specific ionic composition of the external solution. Although ouabain (10(-4)M) alters the pattern of cation accumulation from predominantly potassium to sodium, it does not affect the rate of the reaction, or the total amount of salt or water taken up. To study the response without the complications of active Na-K transport, ouabain was added to most incubations. All water accumulated by the cells can be accounted for by net salt uptake. Specific external cation requirements for reswelling include: sufficient sodium (more than 23 mM), and elevated potassium (more than 7 mM). In the absence of external potassium cells lose potassium without gaining sodium and continue to shrink instead of reswelling. Adding rubidium to the potassium- free solution promotes an even greater loss of cell potassium, yet causes swelling due to a net uptake of sodium and rubidium followed by chloride. The diuretic furosemide (10(-3)M) inhibits net sodium uptake which depends on potassium (or rubidium), as well as inhibits net sodium uptake which depends on sodium. As a result, cell volume is stabilized in the presence of this drug by inhibition of shrinkage, at low, and of swelling at high external potassium. The response has a high apparent energy of activation (15-20 kcal/mol). We propose that net salt and water movements in hypertonic solutions containing ouabain are mediated by direct coupling or cis-interaction, between sodium and potassium so that the uphill movement of one is driven by the downhill movement of the other in the same direction.     

Na(+)-dependent L-arginine transport in the pigmented rabbit conjunctiva

The objective of this study was to characterize Na(+)-coupled L-arginine (L-Arg) transport in the pigmented rabbit conjunctiva. The excised pigmented rabbit conjunctiva was mounted in the modified Ussing chamber for measurement of short-circuit current (Isc), 3H-L-arginine (3H-L-Arg) flux, and 22Na flux. L-Arg when added to the mucosal side led to 0.32-2.65 microA cm-2 increases in the Isc at 37 degrees C, but not at 4 degrees C or in a Na(+)-free solution. L-Arg at 1 mM stimulated net Na+ absorption by 0.12 microEq cm-2 h-1. The evidence for carrier-mediated transport of L-Arg includes: (1) temperature dependence and saturability over 0.01-10 mM, (2) Na+ dependence and ouabain sensitivity, (3) 84 +/- 2% reduction in the apparent permeability coefficient (Papp) of 3H-L-Arg in the presence of excess unlabeled L-Arg (1 mM), and (4) 16-fold difference in L-Arg transport (at 0.1 mM) between the mucosal-to-serosal and the serosal-to-mucosal direction. Moreover, L-Arg transport was inhibited by basic amino acids, large neutral amino acids, and nitric oxide synthase inhibitors, but not by acidic and small neutral amino acids. Kinetic analysis revealed the possible existence of both high and low affinity processes for L-Arg transport. A half maximal concentration (Km) and maximal L-Arg flux (Jmax) values of the low and high affinity processes were 5.90 and 0.07 mM, and 1,248 and 111 pmol cm-2 min-1, respectively. Hill analysis of L-Arg transport at 0.1 mM in the presence of varying Na+ concentrations in the mucosal bathing fluid yielded a Hill coefficient of 0.93, suggesting a 1:1 coupling between Na+ and L-Arg. In conclusion, Na(+)-coupled transport process(es) for L-Arg in accordance with a 1:1 stoichiometry appear to be present on the mucosal side of the pigmented rabbit conjunctiva. The pattern of inhibition by basic and large neutral amino acids and Na+ dependency are suggestive of system B0,(+)-mediated L-Arg transport.     

Swelling-stimulated passive potassium transport in camel erythrocytes: inhibitory effects of furosemide and sodium fluoride

The inhibitory effects of furosemide, sodium fluoride, and age on volume-dependent, ouabain-resistant K+ influx were investigated in camel red blood cells. Swelling of young camel erythrocytes hypotonically stimulates ouabain-resistant potassium influx, a response that was lacking in old camel erythrocytes. The swelling-stimulated influx was partially inhibited by 1 mM furosemide and by 10 and 20 mM sodium fluoride. The inhibitory effect of furosemide was significantly increased if rubidium was added to the flux media. There was a significant correlation between potassium influx in normo- and hypotonic media which might indicate that the anion-dependent transport system operates, to some extent, to regulate cell volume.     

Transport characteristics of the colonic epithelium of the Japanese quail (Coturnix coturnix)

The colon of the domestic fowl sustains a reabsorptive Na+ current on both high- and low-sodium diets. However, there is a marked shift in the apical transport step under these two extreme conditions, from amino acid/hexose cotransport on high-salt diets to amiloride-sensitive Na+ channels on low-salt diets. The present experiments were performed to study colonic Na+ transport in another galliform species, the Japanese quail (Coturnix coturnix). Birds were maintained on a commercial game feed containing 0.18% Na+ (78 mumoles/g), an intermediate level of salt intake. Experiments were performed on unstripped colons in standard Ussing chambers with bicarbonate/CO2 buffer solution on both sides. Baseline values (n = 11) for PD (3.13 +/- 0.68 mV) and short circuit current (SCC, 30.87 +/- 7.79 microA/cm2) were lower than those reported for chickens on a similar diet, whereas tissue resistance (76.06 +/- 4.19 omega.cm2) was similar. Addition of amino acids (4 mM leucine + lysine) increased SCC by 10.85 +/- 1.97 microA/cm2. Both phloridzin (1 mM) and amiloride (10(-5) M) decreased SCC, by 7.05 +/- 1.26 and 9.64 +/- 2.68 microA/cm2, respectively. Thus, on this diet the quail colonic epithelium maintains both amino acid/hexose cotransporter activity and amiloride sensitive channel activity. Arginine vasotocin (10(-6) M) caused a small, but consistent decrease in SCC, while acetazolamide increased SCC. Aldosterone (128 micrograms/kg), given 4 hr prior to the experiment (n = 4) significantly reduced the amino acid stimulated SCC. These results confirm, for the Japanese quail, the presence of multiple apical Na+ entry mechanisms in colonic epithelium. Amino acid cotransporter activity, in particular, appears to be highly sensitive to aldosterone suppression.     

Novel derivatives of cyclodextrins, modified with poly(ethylene oxide) and their complexation properties

Sodium chloride transport across isolated cecum mucosa was investigated in normal rats and rats with adaptive cecum growth induced by dietary polyethylene glycol (PEG). The normal cecum absorbed Cl in excess of Na with a small short-circuit current (ISC). Dietary adaptation led to large equivalent increments of Na and Cl net absorption without adequate ISC change. Inhibitor studies (mucosal amiloride 10(-3) and 10(-4) M; mucosal 4, 4-diisothiocyanatostilbene-2,2-disulfonic acid 5 x 10(-5) M; serosal furosemide 10(-3) M; serosal ouabain 10(-3) M) suggested that normal cecal NaCl absorption involves electroneutral Na/H and Cl/HCO3 exchange at the apical and Na-K-ATPase-mediated exit across the basolateral cell membrane. Dietary adaptation stimulates the loosely coupled antiports and possibly activates a small serosally located NaCl cotransport. Comparative histology showed flattening of all tissue layers and widening of crypts in PEG animals. Crypt widening may facilitate ion access to underutilized transport sites and, at least in part, explain the increased absorption of the enlarged cecum.     

Active sodium-urea counter-transport is inducible in the basolateral membrane of rat renal initial inner medullary collecting ducts

Rat inner medullary collecting ducts (IMCD3s) possess a luminal Na+-dependent, active urea secretory transport process, which is upregulated by water diuresis. In this study of perfused IMCDs microdissected from base (IMCD1), middle (IMCD2), or tip (IMCD3) of the inner medulla, we tested whether furosemide diuresis alters active urea transport. Rats received furosemide (10 mg/d s.c. for 3-4 d) and were compared with pair-fed control rats. Furosemide significantly decreased urine osmolality and urea clearance, and increased blood urea nitrogen. IMCD3s from furosemide-treated rats had significantly lower rates of active urea secretion than IMCD3s from control rats. IMCD2s showed no active urea transport in control or furosemide-treated rats. IMCD1s from control rats had no active urea transport, but IMCD1s from furosemide-treated rats expressed significant rates of active urea reabsorption. In IMCD1s, this active urea reabsorptive transport process was inhibited by: (i) 0. 25 mM phloretin (bath); (ii) 1 mM ouabain (bath); and (iii) replacing bath Na+ with NMDG+; it was stimulated by 10 nM bumetanide (bath). In summary, we found that furosemide decreased active urea secretion in IMCD3s and induced active urea reabsorption in IMCD1s. The new Na+- dependent, active urea reabsorptive transport process may be a basolateral Na+-urea antiporter.     

The effect of transport-blocking drugs on secretion of fluid and electrolytes by the mandibular gland of red kangaroos, Macropus rufus

Mechanisms of primary fluid formation by macropodine mandibular glands were investigated in anaesthetized red kangaroos using ion-transport and carbonic anhydrase inhibitors. Bumetanide at carotid plasma concentrations of 0.005-0.1 mmol/l progressively reduced a stable, acetylcholine-evoked flow rate of 1.02 +/- 0.024 ml/min to 0.16 +/- 0.016 ml/min (mean +/- SEM). Concurrently, saliva [Na], [Cl] and osmolality decreased, [K] and [HCO3] increased and HCO3 excretion was unaffected. High-rate cholinergic stimulation was unable to increase salivary flow above 12 +/- 1.5% of that for equivalent pre-bumetanide stimulation. Furosemide (1.0 mmol/l) and ethacrynate (0.5 mmol/l) caused depression of salivary flow and qualitatively similar effects on ion concentrations to those of bumetanide. Amiloride (up to 0.5 mmol/l) caused no reduction in salivary flow rates or [Na] but decreased [K] and [Cl] and increased [HCO3]. When compared with bumetanide alone, amiloride combined with bumetanide further augmented [K] and [HCO3] and lowered [Cl], but had no additional effects on Na or flow. At the higher level, 4-acetamido-4'- isothiocyanatostilbene-2,2'disulphonic acid (SITS) (0.05 and 0.5 mmol/l) stimulated fluid output, increased [HCO3] and [protein], and depressed [Na], [K] and [Cl]. Relative to bumetanide alone, SITS given with bumetanide had no additional effects on salivary flow or electrolytes. Methazolamide (0.5 mmol/l) in combination with bumetanide curtailed the decrease in [Cl] and the increases in [K] and [HCO3] associated with bumetanide. The residual methazolamide-resistant HCO3 excretion was sufficient to support 2-6% of primary fluid secretion. It was concluded that secretion of primary fluid by the kangaroo mandibular gland is initiated mainly (> 90%) by Cl transport resulting from Na-K-2Cl symport activity. A small proportion of the fluid secretion (up to 6%) appears to be supported by HCO3 secretion. No evidence was found for fluid secretion being dependent on Cl transport involving Na/H and Cl/HCO3 antiports or on HCO3 synthesis involving carbonic anhydrase.     

Active secretion of the fluoroquinolone ciprofloxacin by human intestinal epithelial Caco-2 cell layers

The bidirectional transepithelial fluxes of ciprofloxacin, an antibacterial fluoroquinolone, across the human intestinal epithelial Caco-2 cell-line show marked asymmetry. Basal-to-apical flux of ciprofloxacin (10 microM) exceeds apical-to-basal flux indicating net secretion. Net ciprofloxacin secretion is abolished by azide/2-deoxy-D-glucose treatment, displays saturation kinetics (Km = 0.89 +/- 0.23 mM, Vmax 44.3 +/- 4.9 nmol cm-2.h) and competition by other fluoroquinolones. A specific, active secretion in Caco-2 epithelia may explain the transintestinal elimination of ciprofloxacin observed in pharmacokinetic studies in man.     

Epoxygenase metabolites of arachidonic acid affect electrophysiologic properties of rat tracheal epithelial cells1

Epoxyeicosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids, products of the cytochrome P450 arachidonic acid epoxygenase pathway, have been shown to affect electrolyte transport in the kidney; however, the effects of these compounds on airway epithelial ion transport have not been investigated. Intact rat tracheas and primary cultures of rat tracheal epithelial cells were mounted in Ussing chambers to monitor changes in transepithelial voltage (Vt), short circuit current (Isc) and electrical resistance (Rt), with or without the addition of increasing concentrations (10(-9)-10(-6) M) of arachidonic acid, each of the four regioisomeric EETs and each of the corresponding dihydroxyeicosatrienoic acids. In intact tracheas, 11,12-EET caused dose-dependent decreases in Vt and Isc (DeltaVt = 0. 4 +/- 0.1 mV, DeltaIsc = -16.9 +/- 5.4 microA/cm2 at 10(-6) M, P < . 05 vs. vehicle), whereas changes in Rt were not significantly different than vehicle alone. 11,12-dihydroxyeicosatrienoic acid caused less impressive decreases in Vt and Isc, although arachidonic acid and the other compounds tested were without significant effects. 11,12-EET induced similar changes in cultured tracheal epithelial cell electrical parameters at concentrations as low as 10(-9) M. The effects of 11,12-EET were highly stereoselective, with activity limited to 11(R),12(S)-EET, the least abundant rat lung enantiomer. Pretreatment with amiloride or mucosal exposure to sodium free media did not significantly alter the 11,12-EET-induced changes in Vt. In contrast, pretreatment with bumetanide abolished the 11,12-EET electrophysiologic effects, suggesting that these effects may be mediated through inhibition of a chloride conductive pathway. We conclude that arachidonic acid epoxygenase metabolites cause significant changes in rat airway electrical parameters and may be involved in the control of lung fluid and electrolyte transport.