ATP-regulated chloride conductance in endoplasmic reticulum (ER)-enriched pig pancreas microsomes.

The Cl- conductance of endoplasmic reticulum-enriched pancreatic microsomes was identified. Its regulation by nucleotides was investigated by measuring the rate of cation ionophore-induced microsome swelling in the presence of an inward Cl- gradient. The conductance was solubilized and reconstituted into liposomes. The Cl- conductance in intact microsomes was inhibited by stilbene (10(-4) M) and indanyloxyacetic acid (10(-5) M) derivatives. ATP increased Cl- conductance with half-maximal stimulation at 8 x 10(-6) M. Other trinucleotides (GTP, CTP and UTP) were without effect at 10(-4) M. The non-hydrolysable analogue of ATP, adenosine 5'-[beta gamma-methylene]triphosphate (AppCH2p) increased Cl- conductance with a potency similar to that of ATP. The same concentration of adenosine 5'-[gamma-thio]triphosphate (ATP gamma S) which is a substrate for kinases, had no effect. ATP stimulation of Cl- conductance was inhibited by stilbene derivatives. The data suggest the presence of at least one ATP-binding site, and show that the ATP does not need to be hydrolyzed and that its spatial conformation is important for activating the Cl- conductance. Solubilized microsomal proteins reconstituted into liposomes retained their stilbene-inhibited, ATP-stimulated Cl- conductance. A 167 kDa protein was detected by anti-CFTR antibodies in the intact microsomes, but not in the solubilized proteins. The 64 kDa protein (a component of a ubiquitous Cl- channel) was detected in the both intact and solubilized microsomes. These results suggest that this Cl- conductance is not a CFTR protein.

The effect of a disulfonic acid stilbene on proximal cell membrane potential in Necturus kidney.

The effects of 0.5 mM 4-acetamido-4'-isothiocyano-stilbene-2,2' disulfonic acid on the electrical properties of the peritubular membrane were studied in the proximal tubule of the perfused Necturus kidney. The addition of stilbene isothiocyanate disulfonic acid in peritubular perfusate resulted in an average 4.5 mV hyperpolarization with no detectable changes of peritubular membrane input conductance. The depolarization elicited by high-K media was enhanced by 18% in the presence of stilbene isothiocyanate disulfonic acid, an observation indicating that the inhibitor increased the contribution of potassium to membrane potential, presumably by decreasing anionic permeabilities. The hyperpolarizing effect of stilbene isothiocyanate disulfonic acid was abolished when peritubular bicarbonate was removed from the medium and isoosmotically replaced by chloride. These data suggest that (a) intracellular bicarbonate activity is higher than that predicted from passive distribution, (b) stilbene isothiocyanate disulfonic acid decreases P HCO3, thus hyperpolarizing the membrane, (c) chloride distribution appears to be passive when bicarbonate is removed from the peritubular perfusate. The state of Cl distribution when extracellular bicarbonate is at physiologic concentration cannot be assessed from the present data.

Peculiarities of the Na+/D-glucose cotransport system in Necturus renal tubules.

The effects of D-glucose addition to a glucose-free luminal perfusate were investigated in the proximal tubule of Necturus kidney, by electrophysiological techniques. The main findings are: (1) In the presence of sodium, D-glucose produces 10.5 mV +/- 1.1 (S.E.) depolarization. (2) Phlorizin reduces the magnitude of this response to 2.1 +/- 0.1 mV. (3) The glucose-evoked depolarization, delta VG, does not alter the intracellular K+ activity nor is it affected by peritubular addition of ouabain. (4) Isosmotic reduction of Na+ concentration in luminal perfusate from 95 to 2 mmol/l (choline or Li+ substituting for Na+) does not change the magnitude of delta VG; complete removal of sodium from the lumen lowers the value of delta VG (3.2 +/- 0.2 mV) but the response is not abolished. This observation suggests that the D-glucose carrier of renal tubules in Necturus is poorly specific with regard to the cotransported cation species.

Genome-independent effects of 1,25-dihydroxy vitamin D-3 on membrane potential.

Cell membrane potential, Vm, was monitored in rabbit hypertrophic cartilage metatarsals, amphibian proximal tubule and muscle cells during application of 1,25-dihydroxy vitamin D-3, 25-hydroxy vitamin D-3 or cholesterol (10(-10) M). 1,25-Dihydroxy vitamin D-3 elicited quick variations of Vm (in less than 1 min) in proximal tubular cells (whether injected in the lumen or in peritubular capillaries) and in cartilage. The precursor 25-hydroxy vitamin D-3 and cholesterol produced a small shift of Vm in proximal tubule only when applied from the luminal side, but this change was significantly smaller than that observed with 1,25-dihydroxy vitamin D-3. Muscle cells were unresponsive to both metabolites and cholesterol. It is concluded that rapid effects of 1,25-dihydroxy vitamin D-3 on Vm, in target cells, are specific, most likely due to permeability changes and not related to nuclear protein synthesis; they may contribute to early modulation of cell function.

Conductive properties of the proximal tubule in Necturus kidney.

The electrical properties of the proximal tubule of the in vivo Necturus kidney were investigated by injecting current (as rectangular waves) into the lumen or into the epithelium of single tubules and by studying the resulting changes of transepithelial (VL) and/or cell membrane potential (VC) at various distances from the source. In some experiments paired measurements of VL and VC were performed at two abscissas x and x'. The luminal length constant of about 1,030 micrometer was shown to provide a good estimate of the transepithelial resistance, specific resistance (RTE = 420 omega.cm2) and/or per unit length (rTE = 1.3 x 10(4) omega.cm). The apparent intraepithelial length constant was subject to distortions arising from concomitant current spread in the lumen. The resistances of luminal membrane (rL), basolateral membrane (rB), and shunt pathway (rS) were estimated by two independent methods at 3.5 x 10(4), 1.2 x 10(4), and 1.7 x 10(4) omega.cm, respectively. The corresponding specific resistances were close to 1,200, 600, and 600 omega.cm2. There are two main conclusions of this study. (a) The resistances of cell membranes and shunt pathway are of the same order of magnitude. The figure of the shunt resistance is at variance with the notion that the proximal tubule of Necturus is a leaky epithelium. (b) A rigorous assessment of the conductive properties of concentric cylindrical double cables (such as renal tubules) requires that electrical interactions arising from one cable to another be taken into account. Appropriate equations were developed to deal with this problem.

Actions of isoproterenol in frog proximal tubules.

In the present work, we compared biochemical and electrophysiological actions of isoproterenol on frog proximal tubular cells by using tubule suspensions and giant entities obtained by cell fusion. Isoproterenol (ISO) dose-dependently stimulated cAMP production in tubule suspension and depolarized the "giant cell" membrane. Both effects were triggered by beta receptor occupancy, but strongly differed in their concentration-dependency, since depolarization occurred with an ISO concentration as low as 10(-12) mol/l whereas cAMP accumulation could be seen only with more than 10(-8) mol/l ISO. ISO-induced membrane depolarization was mimicked by forskolin which directly stimulated the catalytic subunit of adenylyl cyclase. In both isoproterenol- and forskolin-stimulated giant cells, membrane depolarization was accompanied by a decrease in membrane conductance, and both effects were inhibited by tetraethylammonium (TEA) and 4-aminopyridine (4-AP). On the other hand, ISO- and forskolin-induced cAMP production were not affected by TEA. The present data thus show that isoproterenol produces two independent effects in frog proximal tubule: it depolarizes the cell membrane by blocking a K+ conductance and activates adenylyl cyclase.

Tunneled double-lumen silicone hemodialysis catheter placement in three patients with permanent pacemaker wires: a case study.

Permanent pacemaker wires have been described as a cause of central vein stenosis. Furthermore, in hemodialysis (HD) patients with transvenous pacemakers, permanent vascular access (VA) created at the ipsilateral arm is not always successful. We report the use of tunneled double-lumen silicone HD catheters, as permanent VA in three HD patients wearing permanent transvenous pacemakers. In one patient, the catheter was inserted ipsilateral to the pacemaker site. Catheter-related infections were the most significant complications.

DNA variation in a 13-Mb region including the F9 gene: inferring the genealogical history and causal role of a hemophilia B mutation (IVS 5+13 A-->G).

About 5.5% of all UK hemophilia B patients have the base substitution IVS 5+13 A-->G as the only change in their factor (F)IX gene (F9). This generates a novel donor splice site which fits the consensus better than the normal intron 5 donor splice. Use of the novel splice site should result in a missense mutation followed by the abnormal addition of four amino acids to the patients' FIX. In order to explain the prevalence of this mutation, its genealogical history is examined. Analysis of restriction fragment length polymorphism in the 21 reference UK individuals (from different families) with the above mutation showed identical haplotypes in 19 while two differed from the rest and from each other. In order to investigate the history of the mutation and to verify that it had occurred independently more than once, the sequence variation in 1.5-kb segments scattered over a 13-Mb region including F9 was examined in 18 patients and 15 controls. This variation was then analyzed with a recently developed Bayesian approach that reconstructs the genealogy of the gene investigated while providing evidence of independent mutations that contribute disconnected branches to the genealogical tree. The method also provides minimum estimates of the age of the mutation inherited by the members of coherent trees. This revealed that 17 or 18 mutant genes descend from a founder who probably lived 450 years ago, while one patient carries an independent mutation. The independent recurrence of the IVS5+13 A-->G mutation strongly supports the conclusion that it is the cause of these patients' mild hemophilia.

Triflocin, a novel inhibitor for the Na-HCO3 symport in the proximal tubule.

1. Triflocin, applied at millimolar concentration hyperpolarizes the basolateral membrane of Necturus proximal convoluted tubular cells, in vivo. 2. Barium, 2.5 x 10(-3) M, ouabain, 10(-3) M, or amiloride 10(-4) M, fail to prevent this hyperpolarization. 3. Triflocin has no effect on the intracellular chloride activity. 4. In physiological acid base conditions, Triflocin increases intracellular pH. 5. Upon an acute isohydric hypercapnia, Triflocin depolarizes the basolateral membrane potential. 6. It is concluded that, Triflocin inhibits the basolateral electrogenic Na-(HCO3)n > 1 cotransport in proximal tubules.

Millimolar amiloride concentrations block K conductance in proximal tubular cells.

1. Amiloride, applied at millimolar concentrations, results in the blockade of K+ conductance in amphibian proximal convoluted cells (PCT), fused into giant cells. 2. Amiloride results directly in a blockade of K+ conductance that is not related to inhibition of the Na(+)-H+ antiport, which would lower intracellular pH, adversely affecting K+ conductance. On the contrary, high amiloride concentrations promote entry of this lipophilic base in the cell, leading to higher cell pH. 3. Under voltage clamp conditions, control vs. amiloride, current-voltage curves from PCT fused giant cells intersect at -86.2 +/- 3.4 mV, a value close to the equilibrium potential for potassium. 4. Hexamethylene amiloride, 10(-5) M, irreversibly depolarizes the membrane potential. 5. Barium decreased by 50% the initial slope of realkalinization, following removal of a solution containing NH4Cl, as did amiloride. In addition, these blockers reduced membrane conductance by 40%, suggesting that a fraction of the amiloride-suppressible NH4+ efflux may be conductive. 6. Amiloride does not directly inhibit the Na(+)-K+, ATPase in our preparation, contrary to the prevalent belief. 7. In vivo studies show that amiloride interferes with an apical K+ conductance but it does not alter basolateral K+ conductance.

The effects of barium on the electrical properties of the basolateral membrane in proximal tubule.

We studied the effects of millimolar Ba2+ concentrations on the properties of the basolateral membrane of the proximal tubule in Necturus kidney. Ba2+ was added in the peritubular perfusate by means of capillary microperfusion experiments. Basolateral membrane p.d. and conductance were continuously monitored in single tubules (their lumen was filled with oil) during reversible application of Ba2+ in peritubular fluid. The effect was membrane depolarization by 14mV and a decrease of membrane conductance to 63% of control values. This association strongly suggests that the main effect of Ba2+ is a decrease of the partial conductance to K+ at the basolateral membrane. However, in other experiments, performed also in oil-filled tubules, Ba2+ was shown to decrease intracellular K+ activity, recorded continuously by means of double-barreled (selective vs non selective) K+ microelectrodes. This and other, indirect evidence support the hypothesis that Ba2+ elicits also a small increase of the partial conductance to sodium at the basolateral membrane.

Opposite modulation of ouabain cardiotoxicity by hexamethyleneamiloride and phenylephrine.

To see whether the Na/H antiporter plays a role in digitalis cardiotoxicity, we investigated the influence of modulators of Na/H exchange on the toxic effects of ouabain in isolated, paced (0.4 Hz) rat left atria. Ouabain (1 mmol/l) caused a transient positive inotropic effect followed by toxic events, including a complete loss of developed force and a gradual increase in resting force. In the presence of hexamethyleneamiloride (3 and 10 mumol/l), an inhibitor of Na/H exchange, ouabain (1 mmol/l) caused a sustained positive inotropic effect without toxicity. By contrast, phenylephrine (100 mumol/l), an alpha-adrenoceptor agonist reported to stimulate the antiporter, hastened the development of ouabain's toxicity. Neither ouabain, at a subtoxic concentration (650 mumol/l), nor phenylephrine (100 mumol/l) affected diastolic force, but in their combined presence, a substantial contracture developed and twitch contractions disappeared. Phenylephrine (30 or 100 mumol/l) or adrenaline (30 mumol/l), in the presence of a beta-adrenoceptor antagonist, increased the intracellular pH by up to 0.15 pH unit, as measured using ion-selective microelectrodes in quiescent preparations. This effect on pHi was prevented by hexamethyleneamiloride (10 mumol/l). Consistent with phenylephrine's ability to stimulate Na+ influx via the Na/H antiporter, phenylephrine (100 mumol/l) increased intracellular Na+ activity by about 3 mmol/l in ouabain (650 mumol/l)-treated atria. These findings indicate that modulators of Na/H exchange affect the cardiotoxicity of digitalis glycosides and imply that the stimulation of myocardial alpha-adrenoceptors may aggravate digitalis toxicity.

Composition, reactivity and surface interactions of three dental silane primers.

Three commercially available dental silane primers, two single-phase prehydrolyzed and one two-component system were investigated for their composition, extent of hydrolysis, surface interactions, bond strength and interfacial topography when used on three representative porcelain alloys (Au-Pd, high-Pd, Ni-Cr). Five tests, 1H FT-NMR, FTIR, GPC, ESCA and EPMA, were used to determine the composition and the surface interaction profiles. Shear tests were performed to assess the bond strength values. According to the results, all the primers contain gamma-methacryloxypropyltrimethoxysilane (gamma-MPTS) at concentrations of 1.15-18.86 g/100 mL, two in ethanol and one in isopropanol. Traces of acetic acid were found in the prehydrolyzed primers. All the primers demonstrated partial hydrolysis of the methoxy groups. Prehydrolyzed primers exhibited a higher rate of hydrolysis and better orientation of hydrolyzed methoxy groups towards the Ni-Cr alloy surface. Due to the extent of surface oxidation, the Ni-Cr alloy provided more bonding sites for silanols than the other two types of alloys. The Ni-Cr alloy demonstrated the highest shear bond strength values on smooth surfaces regardless of the type of primer used. The results of the present study suggest that active prepolymerized primers may provide significant advantages over two-component systems in the repair of Ni-Cr porcelain fractures involving removal of the metal oxide layer.

Experience with the use of uncuffed double-lumen silicone hemodialysis catheters.

AIM: This study aimed to describe our experience with the use of uncuffed double-lumen silicone hemodialysis catheters (USHDCs) that were used in 54 cases as a temporary vascular access (VA). SUBJECTS AND METHODS: We recorded, retrospectively, all the USHDCs (size 13.5 French (F), length 15, 20 and 24 cm) that were inserted in our dialysis unit from July 2003 to September 2004. Catheter and patient characteristics, as well as catheter related complications, were recorded. RESULTS: There were 88 catheters used in 54 cases (44 patients). The catheters remained in place for a total of 2537 days (range 8-127 days, mean 46.9 +/- 31.1). For catheter placement, the internal jugular veins (group A) or the femoral veins (group B) were used in a non-randomized manner. In group A, in 17 cases, 31 catheters were used for a total of 1169 days (mean 68.7 +/- 28.5), while in group B, in 37 cases, 57 catheters were used for a total of 1368 days (mean 36.9 +/- 27.1; p < 0.001). In group B, 81% of cases (30/37) were ambulatory from the time of insertion. Mean urea reduction ratio (URR) in well functioning catheters (blood flow > or = 200 ml/min) was 65.5 +/- 4.6% in group A and 56.9 +/- 6.2% in group B; p < 0.001. Catheter-related bacteremia was observed in five group A cases and in seven group B cases (p = ns). Three cases of minor bleeding at the insertion site and three cases of ipsilateral leg edema were recorded in group B patients. In group A, only one case of bleeding at the insertion site was recorded. CONCLUSION: Uncuffed double-lumen silicone hemodialysis catheters (USHDCs), 13.5 F in size, provided a very efficient temporary VA when placed in the jugular vein. Femoral placement of these catheters can also be used successfully in non bed-ridden patients, but delivering a lower dialysis dose.

Electrophysiological study of the antiluminal membrane in the proximal tubule of Necturus: effect of inorganic anions and SCN-.

1. A study has been made of the effects of anionic substitutions on the electrical potential difference (p.d.) and conductance characteristics of the antiluminal (peritubular) membrane of the proximal tubule of Necturus kidney. The tubular lumina were filled with oil in order to minimize potential and conductance contributions from luminal membrane and from paracellular shunt pathway.2. Isosmotic substitutions, [A](o) for [Cl](o), produced the following average changes in membrane p.d. (mV): F(-) +1.7, BrO(3) (-) +0.1, Br(-) -4.5, ClO(3) (-) -5.2, I(-) -7.9, NO(3) (-) -12.1, ClO(4) (-) -17.8, SCN(-) -25.3.3. The amplitude of the depolarization caused by increase in K concentration (K-depolarization) in the peritubular perfusate was found to increase during perfusion of the tissue with ClO(4) (-) (by 78%), SCN(-) (45%), I(-) (23%), NO(3) (-) (20%), Br(-) (16%); it decreased with F(-) (by 17%).4. Comparison of membrane p.d. at peak K-depolarization in the control state (during KCl perfusion) with that obtained in the experimental state (during KA perfusion) was found to be more reliable than determination of bi-ionic potentials as a qualitative estimate of the permeabilities of the various anions (P(A)) relative to that of chloride (P(Cl)).5. Study of both peak K-depolarization p.d. and bi-ionic potentials yielded the following sequence for halide anion permeabilities: P(F) > P(Cl) > P(Br) > P(I). The peritubular membrane was found to be substantially more permeable to NO(3) (-), ClO(4) (-) and SCN(-) than to Cl(-).6. The sequence of membrane conductances during anionic substitutions was Cl(-) approximately BrO(3) (-) < Br(-)

Identification of membrane transport processes in renal cells, by means of liquid ion exchanger microelectrodes.

The development of liquid-ion-exchanger microelectrodes displaying tip diameters less than or equal to 1 micron has permitted direct measurement of the transmembrane chemical and electrochemical gradients of several permeant ion species in cells of small size. We have used Cl- and H+ resins to study the intracellular Cl- activity (alpha iCl) and cell pH (pHi) in the proximal tubule of Necturus kidney. These determinations were performed in association with perfusion of peritubular capillaries by several artificial solutions, in order to assess the dependence of alpha iCl and pHi on the composition of physiologic plasma constituents and selected inhibitors. The main findings are: Intracellular chloride activity, alpha iCl, is higher than the theoretical value predicted from electrochemical equilibrium. Peritubular application of SITS resulted in a decrease of alpha iCl and increase of pHi; these observations are taken to indicate that Cl- uptake is achieved across the basolateral membrane in exchange for HCO-3 by a mechanism sensitive to SITS. Na+ removal from peritubular fluid elicited a small reduction of alpha iCl, suggesting the presence of carrier-mediated Cl--Na+ cotransport from interstitium to cell, contributing to the rise of alpha iCl above equilibrium. In conclusion, two carrier-mediated processes (Cl-/HCO-3 exchange and Cl--Na+ symport) located at the basolateral membrane of the proximal tubule may account for the establishment of alpha iCl values above equilibrium, at steady state. The physiologic role of these carriers is discussed in relation to proximal electrolyte absorption.

Biionic potentials in the proximal tubule of Necturus kidney.

1. Biionic potentials were studied in the proximal tubule of the doubly perfused Necturus kidney and subsequently analysed by means of an equivalent electrical circuit.2. Luminal membrane resistance was found to be at least 3 times greater than peritubular membrane resistance.3. Potassium contribution to peritubular membrane conductance amounts to at least 75%. Replacement of chloride in peritubular circulation by benzene sulphonate or acetyl glycinate hyperpolarized peritubular membrane.4. The peritubular membrane is more permeable to choline than to sodium. Indirect evidence suggests that the opposite may apply at the luminal border.5. The shunt pathway discriminates poorly, if at all, between sodium, potassium and choline, in short term experiments.6. Chloride permeability across the shunt pathway is about three times as great as combined cationic permeabilities.

Anion permeation in the proximal tubule of Necturus kidney: the shunt pathway.

The effect of foreign anions on transepithelial potential difference and transepithelial input conductance was studied in the isolated perfused Necturus kidney. Two microelectrodes (recording and current-injecting) were inserted into the lumen of single proximal tubules and the peritubular perfusate was shifted reversibly for 30-60 sec from a physiologic Ringer's solution to a test solution in which chloride was replaced isosmotically by a foreign anion. The permeability sequence, obtained by potential measurements, was: lactate less than glutamate less than gluconate less than pyruvate less than benzene sulfonate less than or equal to acetate less than or equal to F less than propionate less than BrO3 less than formate less than ClO3 less than Cl than ClO4 less than I less than or equal to Br less than NO3 less than SCN. Transepithelial conductance decreased when the tissue was perfused with anions less permeable than chloride but the conductance sequence was different from the permeability sequence. Such discrepancies were more pronounced during perfusion with hyperpolarizing anions; ClO4 and I- (both more permeable than chloride) produced an important decrease in transepithelial conductance, followed by incomplete reversibility when the perfusion was shifted again to chloride Ringer's. The results are best explained by the presence of weak positive fixed charges, governing anion permeation, at the shunt pathway of the proximal tubule. An analysis of the data allows tentative estimates of shape and size of the sites.