Impedance analysis in tight epithelia.
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Animals↗
Biomedical subjects
C Clausen
Publications and source records attributed to C Clausen.
Constitutive and transport-related endocytotic pathways in turtle bladder epithelium.
Proton secretion in the urinary bladder of the freshwater turtle is mediated by a proton pump located in the apical membrane of a population of cells characteristically rich in carbonic anhydrase. Earlier studies have demonstrated that these cells exhibit apical-membrane endocytotic and exocytotic processes which are thought to be involved in the regulation of the rate of proton transport via alterations in the number of pumps within the apical membrane. In this study, we sought to characterize these processes using two different methods. Analysis of transepithelial impedance yielded estimates of membrane capacitance which could be related to membrane area, thereby allowing one to monitor net changes in apical-membrane area resulting from changes in the net rates of endo- and exocytosis. Uptake of the fluid-phase marker FITC-dextran provided a measure of net extracellular volume uptake which was related to net rates of endocytosis. Our major conclusions are summarized as follows. The bladder cells exhibit a high baseline rate of endocytosis which appears to be a constitutive process similar to pinocytosis. This process is completely inhibited when ambient temperature is reduced to 15 degrees C. In addition, serosal application of 0.5 mM acetazolamide causes a transient increase in the rate of endocytosis, concomitant with a decrease in the rate of transport. Reduction of ambient temperature to 15 degrees C reduces the rate of acetazolamide-induced endocytosis, but does not abolish it. Addition of 1 mM serosal azide not only prevents the acetazolamide-induced increase in endocytosis, but also prevents the decrease in transport caused by acetazolamide. Azide has no effect on the baseline rate of endocytosis, nor does it prevent inhibition of carbonic anhydrase by acetazolamide. The specificity of azide, coupled with the different temperature sensitivities, demonstrate that the constitutive and transport-dependent endocytotic pathways are distinct processes. The observation that azide prevents both the acetazolamide-induced increase in endocytosis and the decrease in transport strongly supports the notion that endocytosis of proton-pump-containing membrane is requisite for the inhibition of transport by acetazolamide. Finally, the results also demonstrate that acetazolamide does not inhibit proton secretion simply by inhibiting carbonic anhydrase.
Acetazolamide↗
Transport-dependent alterations of membrane properties of mammalian colon measured using impedance analysis.
Direct current (DC) measurement methods have been commonly used to characterize the conductance properties of the mammalian colon. However, these methods provide no information concerning the effects of tissue morphology on the electrophysiological properties of this epithelium. For example, distribution of membrane resistances along narrow fluid-filled spaces such as the lateral intercellular spaces (LIS) or colonic crypts can influence DC measurements of apical and basolateral membrane properties. We used impedance analysis to determine the extent of such distributed resistance effects and to assess the conductance and capacitance properties of the colon. Because capacitance is proportional to membrane area, this method provides new information concerning membrane areas and specific ionic conductances for these membranes. We measured transepithelial impedance under three conditions: control conditions in which the epithelium was open-circuited and bathed on both sides with NaCl-HCO3 Ringer's solutions, amiloride conditions which were similar to control except that 100 microM amiloride was present in the mucosal bathing solution, and mucosal NaCl-free conditions in which mucosal Na and Cl were replaced by potassium and sulfate or gluconate ("K+ Ringer's"). Three morphologically-based equivalent circuit models were used to evaluate the data: a lumped model (which ignores LIS resistance), a LIS distributed model (distributed basolateral membrane impedance) and a crypt-distributed model (distributed apical membrane impedance). To estimate membrane impedances, an independent measurement of paracellular conductance (Gs) was incorporated in the analysis. Although distributed models yielded improved fits of the data, the distributed and lumped models produced similar estimates of membrane parameters. The predicted effects of distributed resistances on DC microelectrode measurements were largest for the LIS-distributed model. LIS-distributed effects would cause a 12-15% underestimate of membrane resistance ratio (Ra/Rb) for the control and amiloride conditions and a 34% underestimate for the "K Ringer's" condition. Distributed resistance effects arising from the crypts would produce a 1-2% overestimate of Ra/Rb. Apical and basolateral membrane impedances differed in the three different experimental conditions. For control conditions, apical membrane capacitance averaged 21 microF/cm2 and the mean apical membrane specific conductance (Ga-norm) was 0.17 mS/microF. The average basolateral membrane capacitance was 11 microF/cm2 with a mean specific conductance (Gb-norm) of 1.27 mS/microF.(ABSTRACT TRUNCATED AT 400 WORDS)
Amiloride↗
In vivo measurement of tubular fluid ferrocyanide with carbon-fiber microelectrodes.
Techniques to construct carbon-fiber microelectrodes and to measure ferrocyanide ion concentration in single nephrons are described. The measurement involves polarizing an inert carbon-fiber microelectrode 500 mV positive with respect to a Ag-AgCl reference, while measuring the faradic current produced by the oxidation of ferrocyanide. A carbon fiber (5-7 micron diam) is heat sealed into a glass micropipette that is then sharpened, silanized, and electrochemically pretreated to minimize electrode degradation by protein. Circuit diagrams for an inexpensive voltage clamp-current monitor and a data sampling device are presented. The electrodes show a linear response to changes in ferrocyanide concentration in large and very small (20 nl) volumes in vitro. The electrodes were used in an electrochemical microassay to determine tubular fluid-to-plasma ferrocyanide concentration ratios and nephron filtration rates with proximal micropuncture samples. The results show excellent agreement with paired determinations using [3H]inulin. In vivo proximal tubule perfusion experiments show a rapid linear response to changes in tubular fluid ferrocyanide concentration. These electrodes permit rapid quantitative measurements of ferrocyanide concentration and water transport in the proximal tubule and may be useful in other biological systems.
Animals↗
Membrane transport parameters in frog corneal epithelium measured using impedance analysis techniques.
Active Cl- transport in bullfrog corneal epithelium was studied using transepithelial impedance analysis methods, and direct-current (DC) measurements of membrane voltages and resistance ratios. The technique allows the estimation of the apical and basolateral membrane conductances, and the paracellular conductance, and does not rely on the use of membrane conductance-altering agents to obtain these measurements as was requisite in earlier DC equivalent-circuit analysis studies. In addition, the analysis results in estimates of the apical and basolateral membrane capacitances, and allows resolution of the paracellular conductance into properties of the tight junctions and lateral spaces. Membrane capacitances (proportional to areas) were used to estimate the specific conductances of the apical and basolateral membranes, as well as to evaluate coupling between the cell layers. We confirm results obtained from earlier studies: apical membrane conductance is proportional to the rate of active Cl- transport and is highly Cl- selective; intracellular Cl- activity is above electrochemical equilibrium, thereby providing a net driving force for apical membrane Cl- exit; the paracellular conductance is comparable to the transcellular conductance. We also found that: the paracellular conductance is composed of the series combination of the junctional conductance and a nonnegligible lateral space resistance; a small K+ conductance reported in the apical membrane may result from Cl- channels possessing a finite permeability to K+; the basolateral membrane areas is 36 times greater than the apical membrane area which is consistent with the notion of electrical coupling between the five to six cell layers of the epithelium; the specific conductance of the basolateral membrane is many times lower than that of the apical membrane; the net transport of Cl- is modulated primarily by changes in the conductance of the apical membrane and not by changes in the net electrochemical gradient resulting from opposite changes in the electrical and chemical gradients; the conductance of the basolateral membrane does not change with transport which implies that the net driving force for K+ exit increases with transport, possibly due to an increase in the intracellular K+ activity.
Animals↗
Membrane electrical parameters in turtle bladder measured using impedance-analysis techniques.
Equivalent-circuit impedance analysis experiments were performed on the urinary bladders of freshwater turtles in order to quantify membrane ionic conductances and areas, and to investigate how changes in these parameters are associated with changes in the rate of proton secretion in this tissue. In all experiments, sodium reabsorption was inhibited thereby unmasking the electrogenic proton secretion process. We report the following: transepithelial impedance is represented exceptionally well by a simple equivalent-circuit model, which results in estimates of the apical and basolateral membrane ionic conductances and capacitances; when sodium transport is inhibited with mucosal amiloride and serosal ouabain, the apical and basolateral membrane conductances and capacitances exhibit a continual decline with time; this decline in the membrane parameters is most likely caused by subtle time-dependent changes in cell volume, resulting in changes in the areas of the apical and basolateral membranes; stable membrane parameters are obtained if the tissue is not treated with ouabain, and if the oncotic pressure of the serosal solution is increased by the addition of 2% albumin; inhibition of proton secretion using acetazolamide in CO2 and HCO3- -free bathing solutions results in a decrease in the area of the apical membrane, with no significant change in its specific conductance; stimulation of proton transport with CO2 and HCO3- -containing serosal solution results in an increase in the apical membrane area and specific conductance. These results show that our methods can be used to measure changes in the membrane electrophysiological parameters that are related to changes in the rate of proton transport. Notably, they can be used to quantify in the live tissue, changes in membrane area resulting from changes in the net rates of endocytosis and exocytosis which are postulated to be intimately involved in the regulation of proton transport.
Animals↗
Proton transport and membrane shuttling in turtle bladder epithelium.
Proton secretion in the urinary bladder of the fresh-water turtle is mediated by proton pumps located in the apical membrane of carbonic-anhydrase (CA)-rich cells. It has been proposed that the rate of proton transport is regulated by endocytotic and exocytotic fusion processes which alter the apical membrane area, and hence number of exposed pumps. Three techniques were used to study this process. Analyses of transepithelial impedance provided estimates of transport-associated changes in net membrane area, as well as other electrical parameters. Electron microscopy allowed visualization of the endocytotic vesicles thought to be involved in the process. Finally, uptake of a fluorescent fluid-phase marker provided measurements of the rates of endocytosis. We report the following: endocytotic and exocytotic processes occur primarily in the CA-rich cells; inhibition of proton transport resulting from 0.5 mM acetazolamide (AZ) results in a decrease in the apical membrane area of approximately 0.47 cm2/cm2 tissue; the apical membrane specific conductance of the CA-rich cells is approximately 220 microS/microF, and possibly represents a Cl- conductance that may function in counter-ion flow; the decline in transport following AZ is not directly proportional to the decline in apical membrane area, suggesting that changes in pump kinetics are also involved in the regulation of transport; the CA-rich cells exhibit a high rate of constitutive pinocytosis, and hence membrane shuttling, which appears to be independent of the rate of transport; AZ induces a transient increase in the rates of endocytosis and shuttling; and the transport-associated changes in apical membrane area may reflect an effect of AZ on a regulated endocytotic pathway which is distinct from the pinocytotic process.
Animals↗
Failure to detect ampicillin-resistant, non-beta-lactamase-producing Haemophilus influenzae by standard disk susceptibility testing.
We questioned whether the apparent rarity of ampicillin-resistant, non-beta-lactamase-producing (NBLP) Haemophilus influenzae was due to failure of detection. We identified ampicillin-resistant and ampicillin-susceptible H. influenzae by the agar dilution technique, using 10(5)- and 10(3)-CFU inocula. We compared the disk susceptibility of 18 ampicillin-resistant NBLP strains, 13 ampicillin-resistant, beta-lactamase-producing strains, and 10 ampicillin-susceptible strains by using standard 10- and 2-micrograms ampicillin disks on two different media. We also investigated the possibility that disks containing 10 micrograms of clavulanic acid and 2 micrograms of ampicillin could be used to distinguish between the two ampicillin-resistant populations. Using the disk containing 2 micrograms of ampicillin, we correctly differentiated all the ampicillin-resistant isolates from the ampicillin-susceptible isolates on both media (a zone diameter of less than or equal to 15 mm was considered resistant). In contrast, the 10-micrograms ampicillin disk failed to detect 44% (8 of 18) of the ampicillin-resistant NBLP strains (a zone diameter of less than or equal to 19 mm was considered resistant). The MIC of ampicillin with the 10(3)-CFU inoculum correlated better with zone diameters than with the 10(5)-CFU inoculum. A difference in zone diameters of greater than or equal to 8 mm between the disk containing 10 micrograms of clavulanic acid and 2 micrograms of ampicillin and the disk containing only 2 micrograms of ampicillin correctly identified all beta-lactamase-producing strains. We conclude that the 2-micrograms ampicillin disk tests more readily identify ampicillin resistance in H. influenzae than do the 10-micrograms ampicillin disk tests. Future investigation should determine whether this in vitro resistance correlates with clinical treatment failures.
Ampicillin↗
[Diagnosis of necrotizing pancreatitis. Comparison between contrast medium-computed tomography and ultrasonics in a clinical study].
Contrast-enhanced computed tomography and (or) sonography was carried out preoperatively in 93 patients undergoing operation for the treatment of severe acute pancreatitis. 77 patients presented with necrotising pancreatitis and 16 with interstitial oedematous pancreatitis predominantly due to biliary causes. The surgical principle involved necrotomy, continuous post-operative bursal lavage or biliary sanitation and (or) pancreas drainage in the case of interstitial oedematous pancreatitis. Sensitivity of contrast-enhanced CT in necrotising pancreatitis was 85%, in localised necrosis 79%, and in extensive necrosis 89.5%. Sonography was negative in 24% of patients and in 37% of those with severe necrotising processes due to extensive masking by intestinal gas. If successful, its sensitivity was 73% only. Thus, sonography is unsuitable for the diagnosis of severe, especially necrotising, pancreatitis; it should be used for confirming the diagnosis and for monitoring the course of mild pancreatitis. Contrast-enhanced CT is indicated for suspected cases involving the necrotising form of the disease, where extensive pancreatic necrosis is registered with high reliability. Controls are advisable for cases with localised pancreatitis.
Acute Disease↗
General method for the derivation and numerical solution of epithelial transport models.
A general method is presented for the formulation and numerical evaluation of mathematical models describing epithelial transport. The method is based on the principles of conservation of mass, and maintenance of electroneutrality within the cells and bathing solutions. It is therefore independent of the specific membrane transport mechanisms, and can be used to evaluate different models describing arbitrary transport processes (including passive, active and cotransport processes). Detailed numerical methods are presented that allow computation of steady-state and transient responses under open-circuit, current-clamp and voltage-clamp conditions, using a general-purpose laboratory minicomputer. To evaluate the utility of this approach, a specific model is presented that is consistent with the Koefoed-Johnson and Ussing hypothesis of sodium transport in tight epithelia (Acta Physiol. Scand. 42:298-308, 1958). This model considers passive transport of an arbitrary number of permeant solutes, active transport of sodium and potassium, and osmotically induced water transport across the apical and basolateral membranes. Results of the model are compared to published experimental measurements in rabbit urinary bladder epithelium.
Animals↗
Lithium concentration in the muscle compartment of manic-depressive patients during lithium therapy.
Pharmacokinetic (PK) techniques were used to study the effect of lithium (Li+) on Li+ fluxes and concentrations in body compartments of manic-depressives. Patients not yet on Li+ therapy were similar to normal controls in all parameters. Comparison of patients before and during chronic Li+ therapy showed no effect of Li+ therapy on intestinal absorption and renal excretion of Li+. The calculated erythrocyte (RBC)-to-plasma Li+ concentration ratio increased with Li+ therapy, as already known from direct measurements. The calculated muscle-to-plasma Li+ concentration ratio was 6-8 times higher than the RBC ratio, and increased from 1.8 to 4.2 with Li+ therapy. The higher Li+ concentration in human muscle compared to RBC is attributed to muscle's higher inside-negative resting potential, and may underlie side effects that arise in muscle from Li+ therapy. The discrepancy between the observed muscle-to-plasma ratio and that predicted for a passively distributed ion is attributed to extrusion by a countertransport process, and the increase in the observed ratio with Li+ therapy is attributed to inhibition of countertransport, as already established for RBC. Since muscle resembles nerve as an excitable cell, muscle Li+ warrants evaluation as a predictor of therapeutic response and side effects during Li+ therapy.
Bipolar Disorder↗
Staff RN: a discharge planner for every patient.
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Humans↗
Muscle lithium in manic-depressive patients during lithium therapy.
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Bipolar Disorder↗
[A randomized in vivo trial of 3 factor VIII preparations].
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Adolescent↗
Use of AC impedance analysis to study membrane changes related to acid secretion in amphibian gastric mucosa.
We have applied transepithelial AC impedance techniques to gastric mucosa to reconcile ultrastructural and electrophysiological findings about gastric acid secretion and the mucosal barrier. By fitting impedance data measured at different HCl secretion rates to equivalent circuit models, we extracted capacitances and resistances (as measures of membrane area and ionic conductance, respectively) for the apical and basolateral membranes. The impedance measurements were found to be incompatible with earlier equivalent circuit models that modeled membrane electrical properties as lumped circuits based on one or two cell types. A distributed circuit model was developed that assumed only one dominant electrical pathway (i.e., one cell type), but that incorporated electrical effects arising from long and narrow membrane-lined structures present in the epithelium (e.g., gastric crypts, tubulovesicles, lateral intercellular spaces). This morphologically based model was found to represent the measured data accurately, and to yield values for membrane capacitances consistent with morphometric measurements of membrane areas. The main physiological conclusions from this analysis were as follows: (a) The dominant transepithelial current pathway may reside in the oxyntic cells. (b) The transepithelial conductance increase associated with the onset of acid secretion is entirely due to increased conductance of the apical membrane. This is in turn due entirely to increased area of this membrane, resulting from incorporation of tubulovesicular membrane. (c) When membrane conductances are normalized to actual membrane area by use of membrane capacitances, it turns out that acid secretion is not associated with a change in specific ionic conductance (change in conductance per unit area) at either the apical or basolateral membrane. (d) The puzzlingly low value of transepithelial resistance (</=400 Omega-cm(2)) arises because there are hundreds or thousands of square centimeters of actual membrane area per square centimeter chamber area. Apical membrane resistance is 25 kOmega-cm(2) (actual membrane area), implying a tight barrier to back-diffusion of protons.
Animals↗
Bacterial localization in patients with end stage renal disease to avoid bilateral nephrectomy before renal transplantation.
Bacterial localization in potential transplant recipients with end stage renal disease and bacteriuria allowed for a significant decrease in the frequency of bilateral nephrectomy before transplantation. Lack of complications after transplantation showed the reliability of preoperative localization, especially if bacteriuria was localized substantially to the bladder despite flank pain and fever.
Adolescent↗
The influence of an angiotensin II antagonist, saralasin, given before donor nephrectomy, on kidney function after transplantation. A controlled prospective study.
Kidneys from cadaver donors who had been treated during the last 10 min before bilateral nephrectomy with an infusion of 10 micrograms/kg-1/min-1 (Sar1, Val5, Ala8)-angiotensin II had a remarkable, significantly lower percentage of acute renal failure (25%) after renal transplantation than untreated kidneys (58.3%). Anuria was seen in the treated group only in 4.2%, although it occurred in the untreated control group in 16.6%. The role of the activated renin-angiotensin system in postischemic acute renal failure is discussed.
Acute Kidney Injury↗
Bulletin of the History of Medicine. Index to volumes and supplements 1933-1982.
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Bibliographies as Topic↗