Negative Potential Difference Research Articles

Perfusion of gills isolated from the hyper-hyporegulating crab Pachygrapsus marmoratus (Crustacea, Decapoda): adaptation of a method.

This paper reports the adaptation of a branchial perfusion technique to the gills of the small hyper-hyporegulating crab, Pachygrapsus marmoratus. The physiological quality of the preparation has been established and preliminary measurements of the transepithelial potential difference (PD) and sodium fluxes were reported. A clear-cut distinction has to be made between anterior and posterior gills. With symmetrical bathing conditions (same saline on both sides of the epithelium), a significant transepithelial PD was measured only in posterior gills isolated from crabs acclimated to dilute sea water. This hemolymph-side negative PD is shown to require biological energy and to be sensitive to ouabain. Na+ influx was larger than efflux, indicating the occurrence of a net inward of Na+. The active nature of the Na+ influx was established.

The amiloride receptor

The diuretics amiloride, triamterene and the spirolactones have the distinct property of reducing kaliuresis while promoting Na + excretion, in contrast to the members of the two other classes of diuretics, the loop diuretics and the thiazides. The common property of the K+-sparing diuretics is to reduce the electrogenic transport of Na + across the socalled "tight" epithelial that line the distal part of the nephron. This property has been studied extensively in experimental models of tight epithelia such as the skin or urinary bladder of amphibian and in perfused collecting tubules of mammalian kidney [4]. While spironolactone acts by inhibiting the complex action of aldosterone on many effector elements (Na,K-ATPase, apical Na + channel, basolateral K + channels, energetic metabolism) it is well known that amiloride produces its effects by inhibiting a highly Na+-selective channel located in the apical membrane of the epithelial cells. How does amiloride prevent the secretion of K + ? Potassium secretion across the apical membrane is driven by the lumen negative potential difference. As the negative intratubular potential is generated by the electrogenic absorption of Na +, it is easy to understand that blocking Na + transport abolishes the driving force for K + secretion. The tight epithelia lining the distal parts of the nephron and of the colon control the final Na + and K + content of the excreta and are therefore the main effector mechanisms by which the organism controls its global Na + and K + balance. Therefore the regulated function of these epithelia is essential for the maintenance of the extracellular fluid volume and blood pressure. This regulation occurs mainly by modulation of the activity of the apical membrane amiloride-sensitive Na + channel, which is the rate-limiting step in transepithelial Na + reabsorption.

Ion transport across the isolated intestinal mucosa of Anguilla anguilla (Pisces)

The posterior intestine of freshwater-adapted Anguilla anguilla has a serosa negative transepithelial potential difference (TPD), and a current corresponding to the flow of negative current towards serosa. The TPD and the short-circuit current (SCC) were inhibited by Na+ and K+ withdrawal, and Cl− substitution or BA2+ addition inverts TPD and SCC, suggesting a Cl− and Na+ current mucosa-serosa, a K+-C1− cotransport and a K+ channel in the mucosal side. The TPD and SCC were inhibited by ouabain, DIDS, furosemide and amiloride, indicating the presence of a Na+-K+-ATPase, and CL−/HCO−3, Na+-K+-Cl− and Na+/H+ cotransporters

Further characterization of the sodium-dependent nucleoside transporter (N3) in choroid plexus from rabbit

The Na +/nucleoside cotransporter in rabbit choroid plexus differs from Na +/nucleoside cotransporters in other tissues in terms of substrate selectivity and stoichiometry. The overall goal of this study was to further characterize the kinetics of this system (N3). Choroid plexus tissue slices obtained from rabbit brain were depleted of ATP and treated with valinomycin and K +. Na +/thymidine uptake at 30 s in the presence of an inside negative potential difference was significantly greater than in the absence of a potential difference. Na +/thymidine uptake was not significantly affected by replacing chloride with either thiocyanate or sulfate. The K m of Na +/guanosine uptake was 149, 85.2 and 30.5 μM in the presence of a 25, 50 and 100 mM Na + gradient, respectively, whereas the V max was unaffected, suggesting that Na + binds first to the cotransporter, then, the nucleoside. Therapeutically relevant base-modified nucleoside analogs, 5-fluorouridine, 2-chloroadenosine and 5-iododeoxyuridine, significantly inhibited Na +/thymidine uptake with IC 50 values ( mean ± S.E. ) of 12.0 ± 2.3, 21.3 ± 2.2 and 24.4 ± 2.1 μ M , respectively, whereas nucleoside analogs structurally modified on the ribose ring, 3′-azidothymidine, dideoxyinosine and dideoxycytidine (100 μM) did not. These studies suggest that Na +/nucleoside cotransport in the choroid plexus is electrogenic and is not dependent on chloride. This cotransporter, which is present in choroid plexus but not in renal brush-border membrane vesicles from rabbit, may play a role in the disposition of clinically relevant base-modified nucleoside analogs into and out of the brain.

Mechanisms behind cyclic changes in duodenal fluid transport in the fasting state.

The relationship between distal duodenal motility, fluid transport and transmural potential difference (PD) was studied in 33 healthy volunteers. Motor activity was registered with a low-compliance pneumohydraulic system; one pressure channel also served as an intraluminal flowing electrode for registration of transmural PD. Net fluid and bicarbonate transport were measured with a triple lumen perfusion technique. The relation between net fluid transport (NFT) and transmural PD depended on the current phase of the migrating motor complex (MMC). In phase I and early phase II (low motor activity), a significant net fluid absorption was seen when the perfusate contained glucose (30 mM). If glucose was substituted with mannitol, NFT was not significantly different from zero and transmural PD was slightly lumen positive. In the early part of the MMC cycle (low motor activity), there was a significant correlation between a more lumen negative transmural PD and a higher net fluid absorption in individual experiments (r = -0.65; P < 0.01). In late phase II of the MMC cycle (high motor activity), NFT changed significantly in the secretory direction in both Krebs mannitol and Krebs glucose perfused segments, and the perfusate-related difference in transport rate was no longer statistically significant. In the Krebs mannitol group, transmural PD became significantly more lumen negative, and there was also a decrease in net bicarbonate absorption as well as a slight acidification of the luminal contents. The relationship between transmural PD and NFT was reversed, i.e. a more lumen negative PD was associated with a higher net fluid secretion in individual experiments (r = 0.82; P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Use of molecular electrostatic potentials for analysis of anticonvulsant activities of phenylsuccinimides.

The present study was performed on a group of 27 derivatives of phenylsuccinimides, of which only 12 were active against maximal electrical shock in spite of the structural similarities of these compounds. The work consisted of four main parts: 1. crystallographic investigations of a subset of chosen compounds; 2. conformational analysis of characteristic molecules from the investigated series, performed by means of molecular mechanics calculations; 3. molecular orbital optimization of all the molecules using the MNDO method starting with conformations obtained in 2; 4. molecular electrostatic potential (MEP) analysis which was performed on the semiempirical (MNDO) and ab initio levels. This research showed that MEP maps provide a signature that distinguishes between active and inactive compounds. There are MEP minima close to the two carbonyl oxygens of the imide ring, and although the magnitude of the difference between the two minima is approximately constant, the sign of the difference provides an activity index. The initial orientations of phenylsuccinimide molecules in relation to the receptor are not equivalent and they depend on the potential distribution around both the succinimide molecules and around the receptor. In the active compounds the negative potential difference at the discussed points most probably influences the initial set-up of the molecules in relation to the receptor and results in a considerably higher probability of the molecules being bound at the right place on the receptor.

Active calcium absorption in primary cultures of cortical collecting duct cells

Primary cultures of rabbit cortical collecting duct (CCD) cells demonstrated accumulation of Ca at the basolateral (BL) side when cultured on either impermeable or permeable supports. Cell monolayers cultured on impermeable plastic surfaces absorbed Ca with such avidity that hydroxyapatite crystals formed. When cultured on a permeable difference. A steady-state BL/A [Ca] ratio of 120 developed across monolayers in 24 h on days 6 through 8 postseeding. Initial rates of unidirectional 45Ca fluxes on days 6 through 8 indicated a negligible BL to A flux (5.4 +/- 2.6 nmol.h-1 x cm-2) compared with A to BL 45Ca flux (99.4 +/- 19.4 nmol.h-1 x cm-2). Parathyroid hormone applied to the BL side had no significant effect on either unidirectional 45Ca flux, but the second messenger analog, 8-bromoadenosine cyclic monophosphate, increased the A to BL flux by 65%. Inhibiting the Na(+)-K+ ATPase with ouabain (10(-4) M) reduced the A to BL flux by 77%; however, a significant net A to BL flux still remained. Apical addition of amiloride (2 x 10(-5) M) did not affect either unidirectional 45Ca flux. In addition, the inorganic Ca channel blockers Ni2+ (100 microM and 1 mM), La3+ (100 microM and 1 mM), and Cd2+ (20 and 50 microM) did not significantly inhibit either unidirectional 45Ca flux. These results demonstrate that CCD monolayers actively absorb Ca and this can be stimulated by cyclic AMP, raising the possibility that apical Ca entry does not involve amiloride-sensitive channels, or typical Ca channels.

Substrate selectivity, potential sensitivity and stoichiometry of Na +-nucleoside transport in brush border membrane vesicles from human kidney

Recently, we demonstrated the presence of a Na +-nucleoside cotransport mechanism that transports both purine and pyrimidine nucleosides in human renal brush-border membrane vesicles (BBMV) (Gutierrez et al. (1992) Biochim. Biophys. Acta 1105, 1–9). The objective of this study was to further elucidate the characteristics of this cotransport system in terms of electrical potential sensitivity, stoichiometry and substrate selectivity with respect to nucleoside analogs. In BBMV from human kidney, Na +-thymidine uptake was stimulated by an inside negative potential difference created by K + and valinomycin. A hyperbolic relationship between initial rate of uridine uptake and Na + concentration was obtained suggesting a Na +-nucleoside coupling stoichiometry of 1:1. Our previous study had demostrated that the pyrimidines, thymidine, cytidine, and uridine and the purines, adenosine, 2′-deoxyadenosine, and guanosine, but not inosine and formycin B, were substrates of this system. To further define the substrate selectivity of the transporter, the interaction of the drugs, 2-chloroadenosine (2-ClAdo), 5-fluorouridine (5-FUrd) and 5-iodo-2′-deoxyuridine (5-IdUrd), nucleoside analogs that are modified on the base moiety was studied. The three compounds inhibited Na +-thymidine uptake in the vesicles via a competitive mechanism. The IC 50 values for 2-ClAdo, 5-FUrd and 5-IdUrd were 75, 49, and 16 μM, respectively. In addition, 5-IdUrd trans-stimulated the initial uptake of thymidine into the vesicles suggesting that the two compounds share the same transporter. Collectively, these data suggest that Na +-nucleoside transport in the human renal brush-border membrane is an electrogenic process and that the kidney may play a role in the disposition and targeting of clinically important nucleoside analogs.

Effect of colcemid on the water permeability response to vasopressin in isolated perfused rabbit collecting tubules.

1. The effect of the microtubule-disruptive agent, colcemid (N-deacetyl-N-methyl-colchicine), on the water permeability response to vasopressin has been investigated in isolated cortical collecting tubules from the rabbit kidney perfused in vitro. 2. Pretreatment of collecting tubules with colcemid inhibited the increase in water permeability elicited by vasopressin, 50 microU ml-1, in a time- and dose-dependent manner. After 75 min exposure to the drug, inhibition of the response to the hormone averaged 72 +/- 6% (n = 4, P < 0.01) at a colcemid concentration of 7.2 x 10(-5) M. Inhibition was estimated to be half-maximal at a colcemid concentration of 1.9 x 10(-6) M. 3. Colcemid, 2.7 x 10(-7) to 7.2 x 10(-5) M, had no effect on basal water permeability nor on the increase in lumen negative potential difference (PD) induced by the hormone. 4. Lumicolcemid, an isomer of colcemid that does not disrupt microtubules, had no influence on the water permeability response to vasopressin. 5. Pretreatment with colcemid, 2.7 x 10(-5) M, for 45 min inhibited the water permeability response to 8-CPT-cAMP, 1.8 x 10(-5) M, by 38 +/- 4% (n = 5, P < 0.01). 6. When collecting tubules were exposed to colcemid, 5.5 x 10(-5) M, for 45 min after the hydrosmotic response to vasopressin had been established, the drug had no influence on the maintenance of the raised water permeability. 7. The results provide further evidence that cytoplasmic microtubules play a role in the initiation of the hydrosmotic response to vasopressin in the mammalian collecting tubule at a site distal to the generation of cyclic AMP.

Characterization of the transport of tri- and dicarboxylates by pig intestinal brush-border membrane vesicles

1. 1. Transport of citrate and fumarate across the pig intestinal brush-border membrane (BBM) was investigated using isolated BBM vesicles. 2. 2. Citrate and fumarate uptake was stimulated by an inwardly directed Na + gradient consistent with Na +/citrate (fumarate) co-transport. Cis-inhibition and Irons -stimulation experiments strongly suggest the existence of a common transport site for tri- and dicarboxylates. 3. 3. The protonated forms of citrate (citrate −1, citrate −2) seem to be much better transported than citrate −3, indicated by the strong stimulation of citrate uptake at an extravesicular pH of 5.6 compared to pH 7.8. 4. 4. Uptake of tri- and dicarboxylates seems to be potential-sensitive since citrate and in particular fumarate transport was enhanced by an inside negative potential difference. 5. 5. Kinetics of succinate transport revealed a single carrier-mediated component with apparent kinetic constants of 0.43 nmol/mg protein-3 s ( V max ) and 0.14 mmol/1 ( K m ).

Chara plasmalemma at high pH: voltage dependence of the conductance at rest and during excitation.

The high pH state of Chara plasmalemma (Bisson, M.A., Walker, N.A. 1980. J. Membrane Biol. 56:1-7) was investigated to obtain detailed current-voltage (I/V) and conductance-voltage (G/V) characteristics in the pH range 7.5 to 12. The resting conductance started to increase at a pH as low as 8.5, doubling at pH 9.5, but the most notable increases occurred between pH 10.5 and 11.5, as observed previously (Bisson, M.A., Walker, N.A. 1980. J. Membrane Biol. 56:1-7; Bisson, M.A., Walker, N.A. 1981. J. Exp. Bot. 32:951-971). The slopes (and shapes) of the I/V curves varied even over minutes, suggesting a shifting population of open channels. Possible contributions of the permeabilities to H+ and OH-, PH and POH, respectively, to the increase in membrane conductance were calculated in the pH range 8.5 to 12. If PH is the main cause for the increase in conductance, it would have to rise by three orders of magnitude between pH 8.5 and 11.5, implying an enormous increase in the open-channel population as pH rises. On the other hand, a comparatively constant POH over that pH range would result in an increase in conductance due to the rise of OH- concentration. This indicates unchanging open-channel population. The transient excitation conductances at pH 7.5 and 11.5 were compared at a range of membrane PD (potential difference) levels. At more positive PD levels (near 0) the transient conductances showed little change as pH was increased. However, near the excitation threshold the conductance at high pH was slower to reach peak and its amplitude was diminished compared to that at neutral pH. This effect was found to be partially due to the pH change itself and partially due to less negative membrane PD at high pH. The changes in excitation transients developed gradually as pH of the medium was increased. These findings are discussed with a recent model of excitation in mind (Shiina, T., Tazawa, M. 1988. J. Membrane Biol. 106:135-139).

In vivo evidence of altered chloride but not potassium secretion in cystic fibrosis rectal mucosa

In cystic fibrosis, cyclic adenosine monophosphate-mediated chloride secretion is abnormal in respiratory, small intestinal, and rectal mucosa. Calcium-mediated chloride secretion is also aberrant in CF small intestinal mucosa in cystic fibrosis, in contrast to the respiratory epithelia, where it appears to be normal. To determine whether this disparity between calcium- and cyclic adenosine monophosphate-mediated chloride secretion exists in cystic fibrosis rectal mucosa in vivo, transrectal potential difference was measured in age-matched adult cystic fibrosis subjects (n = 8) and control subjects (n = 9) in response to 10-minute luminal perfusions of bethanechol (1 mmol/L) or theophylline (5 mmol/ L). In response to bethanechol, an initial (1-minute) negative change in potential difference (−1.4 ± 1.1 mV; mean ± SEM) was seen in control subjects, in contrast to a positive change in mean potential difference (+2.5 ± 1.0 mV) in cystic fibrosis subjects (control vs. cystic fibrosis, P < 0.05). After 1 minute, mean potential difference changes in both control and cystic fibrosis subjects were positive. Theophylline perfusion resulted in a significant (P < 0.01) difference in potential difference response between groups; at 10 minutes, the potential difference became more negative (−3.6 ± 1.4 mV) in control subjects and more positive in cystic fibrosis subjects (+3.9 ± 1.4 mV). To determine whether second messenger-mediated potassium secretion contributed to the observed potential difference changes in response to bethanechol and theophylline, studies were repeated in the presence of barium chloride, a known blocker of potassium conductance. In the control group, barium chloride significantly enhanced the theophylline-induced negative potential difference change (P < 0.05) and reduced the positive potential difference change seen with bethanechol alone. In subjects with cystic fibrosis, barium chloride completely abolished the previously seen positive potential difference change in response to either bethanechol or theophylline alone. These in vivo studies suggest that there is active potassium secretion in both control and cystic fibrosis rectal mucosa in response to cyclic adenosine monophosphate- and calcium-dependent secretagogues and that the magnitude of the potential difference changes attributable to barium-inhibitable potassium secretion is the same in cystic fibrosis and control subjects. In contrast, it appears that in cystic fibrosis rectal mucosa in vivo, calcium- as well as cyclic adenosine monophosphate-dependent chloride secretion is aberrant.

Characterization of a mouse cortical collecting duct cell line.

A cortical collecting duct (CCD) cell line has been developed from a mouse transgenic for the early region of simian virus 40, Tg(SV40E)Bri/7. CCDs were microdissected and placed on collagen gels. Monolayers were subsequently subcultured onto permeable collagen membranes and maintained in serum-supplemented medium. One line, designated M-1, retained many characteristics of the CCD, including a typical epithelial appearance and CCD-specific antigens. M-1 cells, when grown in monolayers on permeable supports, exhibited a high transepithelial resistance (885.7 +/- 109.6 ohms/cm2) and developed a lumen negative transepithelial potential difference (PD) of -45.7 +/- 3.5 mV. The associated short-circuit current (SCC) averaged 71.8 +/- 10.3 microA/cm2, and was reduced by 95% by luminal application of amiloride. The cultured cells responded to arginine vasopressin (AVP) with a significant increase in SCC. M-1 cells generated significant transepithelial solute gradients. After 24 hours incubation, the composition of the luminal (L) and basolateral (B) media (in mM) was: [Na+], L = 106.7 +/- 0.9 and B = 127.4 +/- 0.4; [K+], L = 8.6 +/- 0.6 and B = 2.1 +/- 0.3; [Cl], L = 68.6 +/- 5.8 and B = 101.8 +/- 6.6; [HCO3], L = 15.5 +/- 1.5 and B = 8.6 +/- 1.2; while pH was 7.16 +/- 0.03 at the luminal and 6.94 +/- 0.03 at the basolateral side. The formation of these concentration gradients indicates that the CCD cultures absorb Na+ and Cl- and secrete K+.(ABSTRACT TRUNCATED AT 250 WORDS)

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

1. 1. Prairie dog gallbladders mounted in a Ussing-type chamber and bathed with symmetrical Ringer's solutions exhibited a transepithelial resistance ( R t ) of 51 ± 51Ω cm 2 , a lumen negative potential difference (V ms) of 11.5 ± 0.7 mV and a short-circuit current (I sc) of 6.9 ± 0.3 μEq/hr/cm 2. 2. 2. Radioisotopic ion flux experiments revealed that the basal I sc of 6.9 ± 0.3 μEq/hr/cm 2 was mostly accounted for by net Na + absorption of 3.2 ± 0.5 μEq/hr/cm 2 and net Cl − secretion of 2.9 ± 0.3 μEq/hr/cm 2. 3. 3. In HCO 3 − free Ringer's, net Na + flux was virtually abolished, net Cl − flux decreased by 50% and I sc was reduced by 77%. 4. 4. 10 −3 M mucosal amiloride and DIDS reduced I sc by 28 and 24%, respectively. 5. 5. Mucosal NaCl diffusion potentials indicated that the paracellular pathway was cation selective. 6. 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. 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 −.

Nasal potential difference: a clinical diagnostic test for cystic fibrosis

Patients with cystic fibrosis (CF) demonstrate a markedly more negative potential difference (PD) across respiratory epithelia than normal or "diseased" controls. A technique is described for the measurement of nasal PD in both children and adults. 145 non-CF subjects showed a mean PD of -19.0 mV (range -2 to -36) in comparison to 60 patients with cystic fibrosis with mean of -46.0 mV (range -32 to -77). Amongst the latter group those with more severe disease had a more negative PD. Measurement of nasal PD is easily learnt and rapidly performed and may provide an additional means of diagnosis for CF.

Atrial peptide and cGMP effects on NaCl transport in inner medullary collecting duct

We examined the action of atrial natriuretic factor (ANF) on Na+ and Cl- transport in in vitro microperfused inner medullary collecting ducts (IMCD) isolated from rat kidneys. First we studied the isotopic fluxes at low perfusion rates (7 nl/min). The results showed that ANF added to bath decreased lumen-to-bath flux (Jl----b) of Na+ and increased Na+ bath-to-lumen flux (Jb----l). This was substantiated by a direct demonstration that ANF reduces net Na+ and Cl- absorption. The effect of ANF on Jl----b and Jb----l of Na+ was also observed at high perfusion rates (25 nl/min). The inhibitory effect of ANF was observed even when Na+ Jl----b was stimulated by vasopressin (VP). ANF (6 x 10(-11) M) added to bath increased Cl- Jb----l and generated a negative lumen potential difference (PD). These two effects were inhibited by furosemide and by the replacement of Na+ by choline and Cl- by SO4(2-) in the bath fluid. These observations are compatible with the existence of a Na(+)-Cl(-)-K+ cotransport mechanism stimulated by ANF. Moreover, the effects of guanosine 3',5'-cyclic monophosphate (cGMP) added to the bath on PD, Jl----b, and Jb----l of Na+ were similar to those observed with ANF. Thus, physiological concentrations of ANF inhibit directly Na+ and Cl- absorption in IMCD by two mechanisms, 1) by increasing cotransport Na(+)-Cl(-)-K+ secretion and 2) by inhibiting NaCl absorption both in the absence and in the presence of VP. These effects on NaCl transport appear to be mediated by cGMP.

Effects of digitoxin and lithium, used as a marker of passive Na transport, on secretin‐dependent bile flow in the pig

The present study was performed in anaesthetized pigs, and the first aim was to assess the role of Na,K-ATPase in secretin-dependent biliary HCO3 secretion (JbHCO3). Intra-arterial administration of the cardiac glycoside digitoxin (0.2 mg/kg-1) reduced hepatic Na K-ATPase activity, JbHCO3 and secretin-dependent bile flow by 24, 55 and 34% respectively. In the second part of this study lithium (Li) was used as a marker of passive Na transport to assess the electrochemical gradient for Na flux into bile duct lumen during secretin-stimulated bile flow and impeded biliary osmotic water flow by i.v. infusion of glucose. At plasma glucose 85 (73-96) mmol l-1, bile [Na] and [Li] exceeded their concentrations in plasma by 57 and 47% respectively. By using the Nernst equation, transepithelial potential difference (PD) during hyperglycaemia was estimated to be -6.2 (0 to -10.8) mV (ductal lumen negative), which corresponds to a [Li]bile/[Li]plasma ratio of 1.3 (1.0-1.5). The ratio was not significantly different from the observed [Li]bile/[Li]plasma ratio of 1.4 (1.3-1.5). It is concluded (1) that Na, K-ATPase is necessary for JbHCO3, probably by sustaining the cell membrane PD (cell interior negative) which is a driving force for apical electrogenic HCO3 secretion, and (2) transepithelial Li (and hence Na) flux is driven solely by the negative transcellular PD during secretin-stimulated bile flow in the pig.

Conductive sodium entry in gill cells of the shore crab, Carcinus maenas

Isolated posterior gills of shore crabs, Carcinus maenas, collected from the Baltic Sea, were perfused and bathed with sea water and solutions of alkali chlorides. The preparation was used to measure fluxes of sodium from the external medium across the gills into the hemolymph and to determine transepithelial potential differences (PDs). Internally negative active transport PDs resulted from perfusion and bathing the gills symmetrically with the same medium (50% sea water). Passive (asymmetry) PDs following employment of 100% sea water as internal and 50% sea water as external medium were — in contrast to symmetry PDs — insensitive to cyanide and ouabain. This result indicates that the gill recognized the desired hyperosmotic state and responded by switching off the active transport component observed under symmetry conditions. Diffusional potential differences and fluxes of Na+ were inhibited by the externally applied diuretic amiloride. Gradients of pure alkali chlorides between medium and blood were accompanied by cation specific PDs. These PDs and their amiloride sensitivity were inversely related to the diameter of the unhydrated cation and allowed the calculation of the permeability sequence P: Li+>Na+>K+>Rb+>Cs+>Cl-. The results obtained show that the permeability of the gills to cations greatly exceeds that to anions. In addition, these findings indicate that the initial amiloride-sensitive step in Na+ transport across the gill is not represented by an electroneutral sodium/proton exchange but by a conductive mechanism.

The effect of parathyroid hormone on cisplatin nephrotoxicity.

The clinical use of cisplatin as an antineoplastic agent is limited by its nephrotoxicity that, in many cases, includes acute tubular necrosis and renal wasting of various electrolytes (1–4). Several studies have been performed to account for the various disorders of renal function induced by cisplatin. The decrease in GFR, for example, has been found to be related to a reduction in renal blood flow and to a lowered effective filtration pressure (5). The reported concentrating defect and the observed impaired sodium reabsorption have been associated to a defect in papillary hypertonicity found in cisplatin treated rats (6), while the potassium wasting effect is in part due to a negative potential difference induced by cisplatin in late distal segments (6). Finally the pathogenesis of cisplatin induced hypomagnesemia has been attributed to pathological changes confined to the straight portion (S3 segment) of superficial nephrons (7). On the other hand many therapeutic strategies have been tested to reduce its nephrotoxic action: the fall in GFR, for example, can be modified by hydration, mannitol diuresis (8) and by the administration of atrial natriuretic peptide (9); moreover compounds that provide SH groups have been reported to reduce the cisplatin associated renal injury (10).KeywordsAcute Renal FailureAtrial Natriuretic PeptidePlasma Calcium LevelRenal Cortical SliceSuperficial NephronThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Primary liver cell cultures grown on gas permeable membrane as source for the collection of primary bile

Isolated rat hepatocytes maintained in primary culture on gas permeable membrane for 20 h form monolayers and establish at their cell borders a network of canaliculi (approximate diameter 3.5 micron). In the presence of the known choleretic bile acid dehydrocholate, dilation of canaliculi occurs. When nonfluorescent carboxyfluorescein diacetate ester is added to the culture medium, fluorescent carboxyfluorescein appears in the intracanalicular space. In the dilated state, fluid containing the fluorescent compound could be collected from the canaliculi by puncture with a micropipette. The intracanalicular space shows a negative electrical potential difference of 31 mV in reference to the bath solution and is 13.5 mV more positive with reference to recordings from the cytosol of cultured rat hepatocytes. Cultured rat hepatocytes grown on gas permeable membrane are energetically stable over 3 d. On Day 4, ATP levels increase markedly, whereas Na+-K+-ATPase activity declines. Ionic composition of hepatocytes, as measured by electronprobe element analysis on cryosection samples, does not change markedly during monolayer formation. With formation of bile canaliculi, the activity of alkaline phosphatase rapidly increases within 24 h and is stable for the next 3 d. Within that time the activity of gamma-glutamyltranspeptidase, however, increases steadily, reaching a 1.6-fold higher activity than freshly isolated hepatocytes. Bile acids appear in the culture supernatant after 1 d. When unconjugated [14C]cholic acid is added to the cultures the supernatant contains also [14C]tauro- and [14C]glycocholic acid, indicating the preservation of conjugation capacity in these cultures. Total bile acid concentrations in the supernatant increase from 5 to 26 microM on Day 4. The cultures do not secrete alpha-fetoprotein. Monolayer cultures of hepatocytes in the presence of choleretic bile acids seem to be a suitable model system to collect and to analyze the composition of primary bile. In conjunction with the electrical parameters, it is possible to describe directly properties of bile secretion at the canalicular pole of the intact hepatocyte.